Smart nucleic acid chaperones

Abstract

The described technology pertains to smart nucleic acid chaperones (SNACs) designed to improve nucleic acid hybridization and assembly processes.

Description

[0001] SMART NUCLEIC ACID CHAPERONES

[0002] PRIORITY

[0003] This application claims the benefit of the filing date of U.S. provisional application No. 63 / 729,199, filed December 6, 2024, the disclosure of which is incorporated by reference herein in its entirety.

[0004] GOVERNMENT GRANT SUPPORT

[0005] This invention was made with government support under 2031774, 2048051, 2123465, 2318897 and 2421209 awarded by the National Science Foundation. The government has certain rights in the invention.

[0006] FIELD OF INVENTION

[0007] The present disclosure pertains to the field of molecular biology' and nucleic acid nanotechnology, specifically addressing smart nucleic acid chaperones (SNACs) that facilitate nucleic acid hybridization and assembly processes through liquid-liquid phase separation (LLPS).

[0008] BACKGROUND

[0009] Nucleic acid (NA) hybridization reactions are fundamental to molecular biology and nucleic acid nanotechnology. These reactions take advantage of the programmable and predictable nature of base-pairing to generate complex structures and dynamic, responsive reaction systems. However, many NA hybridization reactions face significant kinetic barriers that can limit their effectiveness.

[0010] In biology, proteins called nucleic acid chaperones assist in structural changes of macromolecules and influence the kinetics of molecular reactions. DNA and RNA binding proteins are associated with regulating genomic functions through both specific and promiscuous interactions. Among these DNA / RNA binding proteins, intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) of proteins can play significant roles in nucleic acid binding.

[0011] IDPs are proteins that lack a well-defined three-dimensional structure, breaking the traditional paradigm of structure-protein function relationships. Despite their disordered nature, IDPs are involved in crucial biological processes including cell signaling and viral packaging. One family of synthetic IDPs, called elastin-like polypeptides (ELPs), consists of repetitive sequences that can undergo reversible liquid-liquid phase separation (LLPS) to form protein-rich condensates called coacervates.

[0012] Current approaches to enhancing NA hybridization reactions include increasing reactant concentrations or using protein chaperones. Protein chaperoning of complementary DNA hybridization can occur through mechanisms related to structural changes via folding / unfolding or through electrostatic interactions that bring together complementary sequences. Several viral capsid proteins, such as the hepatitis C virus core protein, have been found useful in chaperoning DNA strand annealing and displacement reactions.

[0013] SUMMARY OF THE INVENTION

[0014] One embodiment provides a method to increase nucleic acid hybridization comprising: a) contacting complementary nucleic acid strands with at least one smart nucleic acid chaperone (SNAC), wherein the SNAC comprises a nucleic acid chaperone polymer that undergoes liquid-liquid phase separation (LLPS); b) allowing the at least one SNAC to form a coacervate, encapsulate nucleic acids and chaperone hybridization of the complementary strands, wherein hybridization kinetics are increased as compared to hybridization kinetics without the presence of the at least one SNAC. In one embodiment, the polymer comprises a synthetic polymer, elastin-like polypeptide (ELP), elastin, resilin, RGG, and HCV core protein (HCV CP), or a combination thereof. In one embodiment, the ELP comprises repetitive sequences of V-P-G-X-G (SEQ ID NO: 1), where X is any amino acid except proline. In one embodiment, the ELP comprises SEQ ID NO: 11 (E3), SEQ ID NO: 16, SEQ ID NO: 30 (H24), a combination thereof or 95% identity thereto. In one embodiment, the SNAC comprises a nucleic acid binding domain, a viral capsid protein or combination thereof. In one embodiment, the nucleic acid binding domain comprises an RNA recognition motif (RRM) and / or an arginine-glycine (RGG) domain and / or the viral capsid protein comprises a hepatitis C virus core protein (HCV CP). In one embodiment, the complementary nucleic acid strands comprise self-complementary regions that form hairpin structures. In one embodiment, the complementary nucleic acid strands comprise toehold regions for strand displacement reactions. In one embodiment, b) further comprises inducing liquid-liquid phase separation (LLPS) of the SNAC to form protein-rich coacervates that concentrate the nucleic acid strands, wherein the protein-rich coacervates form nucleoprotein condensates that speed up hybridization kinetics. In one embodiment, the protein-rich coacervates enhance the rate of hybridization up to about 3- to about 60-fold (such as about 3- to about 10-fold, about 10- to about 20-fold, about 20- to about 30-fold, about 30- to about 40-f old, about 40- to about 50- fold, about 50- to about 60-fold, including, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60 fold increase, including about 54-fold increase) compared to hybridization without coacervates.

[0015] One embodiment provides a method to increase hybridization of nucleic acids comprising: a) contacting complementary nucleic acid strands with at least one pH-switchable smart nucleic acid chaperone (SNAC) polymer at a first pH, wherein the SNAC comprises a nucleic acid chaperone polymer that undergoes liquid-liquid phase separation (LLPS); b) allowing the at least one SNAC to form a coacervate, encapsulate nucleic acids and chaperone hybridization of the complementary nucleic acid strands of a); and c) after b) adjusting to a second pH to release the at least one SNAC from the hybridized nucleic acids, wherein hybridization kinetics are increased as compared to hybridization kinetics without the presence of the at least one SNAC. In one embodiment, the polymer comprises a synthetic polymer, elastin-like polypeptide (ELP), elastin, resilin, RGG, and HCV core protein (HCV CP), or a combination thereof. In one embodiment, the at least one SNAC comprises SEQ ID NO: 29, SEQ ID NO: 30 or 95% identity thereto. In one embodiment, the first pH is below the isoelectric point and the second pH is above the isoelectric point of the SNAC. In one embodiment, the first pH is about 6 to 8 (including about 6.0, about 6. 1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8. about 6.9, about 7.0. about 7. 1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 , about 8.2, about 8.3, about 8.4) and the second pH is about 8.5 to 9.0 (including about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0). In one embodiment, in b) protein / nucleic acid-containing coacervates are formed in which the SNAC chaperones hybridization of the complementary nucleic acid strands within the coacervates, wherein the protein / nucleic acidcontaining coacervates form nucleoprotein condensates that speed up hybridization kinetics. In one embodiment, in c) the assembled components from the coacervates of b) are released and the chaperoning activity is disrupted, and the chaperone and nucleic acid product are separated. In one embodiment, the complementary nucleic acid strands comprise self-complementary regions that form hairpin structures. In one embodiment, the complementary nucleic acid strands comprise toehold regions for strand displacement reactions. In one embodiment, the protein-rich coacervates enhance the rate of hybridization up to 3- to 60-fold (and value or range therein) compared to hybridization without coacervates.

[0016] One embodiment provides method to increase hybridization of nucleic acids comprising: a) contacting complementary nucleic acid strands with at least one smart nucleic acid chaperone (SNAC) polymer at a first temperature, wherein the SNAC comprises a nucleic acid chaperone polymer that undergoes liquid-liquid phase separation (LLPS); b) allowing the SNAC to form a coacervate, encapsulate nucleic acids and chaperone hybridization of the complementary nucleic acid strands: and c) adjusting to a second temperature to dissolve the coacerv ate and release the SNAC from the hybridized nucleic acids, wherein hybridization kinetics are increased as compared to hybridization kinetics without the presence of the at least one SNAC In one embodiment, the polymer comprises a synthetic polymer, elastin-like polypeptide (ELP), elastin, resilin, RGG, and HCV core protein (HCV CP), or a combination thereof. In one embodiment, the first temperature is above the SNAC / IDP transition temperature, and the second temperature is below the SNAC / IDP transition temperature (such as above about 30°C, or between about 30°C to about 60°C, depending on the SNAC / ELP / IDP), and below about 30°C, or between about 5°C to about 30°C) depending on the SNAC / ELP / IDP). In one embodiment, in b) protein / nucleic acid-containing coacervates are formed in which the SNAC chaperones hybridization of the complementary nucleic acid strands within the coacervates, wherein the protein / nucleic acid-containing coacervates form nucleoprotein condensates that speed up hybridization kinetics. In one embodiment, liquidliquid phase separation (LLPS) is induced at the first temperature to form the coacervates and enhance hybridization. In one embodiment, in c) the assembled components from the coacerv ates of are released and the chaperoning activity is disrupted. In one embodiment, the temperature is lowered to dissolve the coacervates to release the hybridized products. In one embodiment, the IDP comprises elastin-like polypeptide (ELP), collagen, elastin, resilin, RRM-RGG, and EICV core protein, or a combination thereof. In one embodiment, the ELP comprises SEQ ID NO: 11 (E3), SEQ ID NO: 16, SEQ ID NO: 30 (H24), a combination thereof or 95% identity thereto. In one embodiment, the complementary nucleic acid strands comprise self-complementary regions that form hairpin structures. In one embodiment, the complementary nucleic acid strands comprise toehold regions for strand displacement reactions. In one embodiment, the protein-rich coacervates enhance the rate of hybridization up to about 3- to about 60-fold (or any value or range within / inclusive) compared to hybridization without coacervates.

[0017] One embodiment provides a composition comprising: complementary nucleic acid strands; and i) at least one smart nucleic acid chaperone (SNAC), wherein the SNAC comprises a nucleic acid chaperone protein; and an intrinsically disordered protein (IDP), wherein the nucleic acid chaperone protein and IDP are fused together to form a fusion protein; or ii) at least one intrinsically disordered protein (IDP), wherein the IDP is capable of chaperoning nucleic acids without a fused protein; wherein upon liquid-liquid phase separation (LLPS), the SNAC forms protein containing coacervates that recruit and concentrate the nucleic acid strands, wherein the complementary nucleic acid strands comprise self-complementary regions that form hairpin structures and / or wherein the complementary nucleic acid strands comprise toehold regions for strand displacement reactions. In one embodiment, the SNAC is a pH- switchable SNAC. In one embodiment, the IDP comprises elastin-like polypeptide (ELP), collagen, elastin, resilin, RRM-RGG, and HCV core protein, or a combination thereof. In one embodiment, the ELP comprises SEQ ID NO: 11 (E3). SEQ ID NO: 16, SEQ ID NO: 30 (H24), a combination thereof or 95% identity thereto.

[0018] BRIEF DESCRIPTION OF THE FIGURES

[0019] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0020] FIGS. 1A-1D. Concentration dependent binding of E3. 10 and El-40. COR30 to ssDNA and dsDNA. 2.5% agarose gels stained with SyBr Gold that illustrate the concentrationdependent binding activity E3.10 and El-40. COR30 with (A,C) O. lpM ssDNA Tar(+) and (B,D) O. lpM dsDNA Tar(+»-). E1-40.COR30 concentrations are: 0.1, 0.5, 1,5, 7.5, 10, 25, 50, 75, 100, 250, 500, lOOOpM; E3.10 concentrations are: 0.001, 0.1, 0.5, 1,2.5, 5, 7.5, 10, 25, 50, 75, 100, 150. 200pM.

[0021] FIGS. 2A-2D. Concentration-dependent chaperoning of strand annealing reaction by E1-40.COR30 and E3. 10. (A) Scheme for the SA of 56 nt oligos, Tar (-) and Tar (+) at 25°C. (B) Scheme for the SA of 56 nt oligos, Tar (-) and Tar (+) at 62°C (C) 2.5% agarose gels stained with SyBr Gold show that the annealing of complementary 56 nt strands of 0. IpM Tar (-) and O.lpM Tar (+) is chaperoned by E1-40.COR30. Both oligos are incubated for 10 min at room temperature with an increasing concentration of protein. Protein concentrations are 0.001, 0.01, 0.1, 1, 10, 50, 75, 100, 250, 500 and 1000 pM. (D) Same conditions as in (B) with E3.10. Protein concentrations are 0.001, 0.005, 0.01, 0.05, 0.1, 1, 5 and 10, 50, 100 and 200 pM.

[0022] FIGS. 3A-3B. Kinetics of strand annealing in the presence of soluble SNACS (25°C). (A) Scheme for the fluorescence quenching assay for annealing at 25°C of the modified 56 nt oligonucleotides, F-Tar (-) and Q-Tar (+), which are complementary over 51 nt. (B) Fluorescence-quenching assays to observe the real-time annealing of 0. 1 pM 5IABkFQ -labeled Q-Tar (+) and 0.1 pM 3ATTO488-labeled F-Tar (-) at 25°C in the absence and presence of protein. Protein concentrations are 10 pM for E3.10, 75 pM for E1-40.COR30 and 2mM for E3. Duplex formation is the normalized fraction of F-Tar (-) annealed to Q-Tar (+) (see Methods for normalization equation). Error bars represent the standard deviation of our measurements in triplicate.

[0023] FIGS. 4A-4C. Effect of DNA on LLPS of SNACS and chaperoning of SA reaction inside a protein-rich condensate upon phase separation of ELPs. (A) Scheme for the fluorescence quenching assay for annealing, at 45°C inside an ELP coacervate upon LLPS, of the modified 56 nt oligonucleotides, F-Tar (-) and Q-Tar (+), which are complementary' over 51 nt. (B) Turbidimetry measurements of LLPS Tt of 1.5mM E3, 0.5mM E1-40.COR30, and lOpM E3.10 with and without O. lpM Tar (+). (C) Fluorescence-quenching assay to observe the kinetics of annealing of O. l pM lABkFQ -labeled Q-Tar (+) and O. l pM ATTO488-labeled F-Tar (-) at 45°C in the absence and in the presence of protein. At 45°C, the proteins and oligos form a phase-separated nucleoprotein coacervate. Protein concentrations are 10 pM for E3. 10, 500 pM for E1-40.COR30 and 1.5mM for E3. Extent of the reaction is the fraction of F-Tar (- ) annealed to Q-Tar (+). Error bars represent the standard deviation of our measurements in triplicate.

[0024] FIG. 5. Predicted structure of DNA oligos. The putative predicted structure obtained by NuPack41at 25°C and 45°C of all the oligos used in SA.

[0025] FIG. 6. DNA binding activity of soluble E3. 2.5% agarose gels stained with SyBr Gold illustrate the lack of binding of 0.5pM Tar (+) to E3 over a range of concentrations. (E3 concentrations are: 10, 100, 1000 pM.) FIG. 7. panels Al-E. Raw data for fluorimetry measurements of SA reactions. Part of the raw data from ATTO488 RFU. from F-Tar (-). emission overtime used to obtain the duplex formation of SA reactions at 25°C and 45°C. Error bars represent the standard deviation of our measurements in triplicate. (Q-Tar(+) signal was plotted only in (Al) as a visual reference but not in the rest so as to not overlay with annealing reactions.)

[0026] FIG. 8, panels A1-H4. SA at 25°C under fluorescence microscopy. Polydisperse micro drops showing the SA annealing fluorescent quenching reactions under a fluorescence microscope. See text for details. Scale bar = 50pm.

[0027] FIGS. 9A-9C. Temperature-dependent absorbance measurements (Raw Data). The results from replicate measurements of (A) 1.5mM E3, (B) 0.5mM E1-40.COR30 and (C) lOpM by temperature-dependent absorbance measurements at 380nm are presented without ssDNA and with ssDNA.

[0028] FIGS. 10, panels A1-F4. Photomicrographs for 0.1 pM pM F-Tar (-) with different proteins over a range of temperatures. Representative images of one droplet in brightfield (D- F) and fluorescence emission (A-C). The brightfield images show ELP coacervation while the fluorescence images show the behavior of F-Tar(-) as time and temperature increase (left to right). Scale bar = 50pm.

[0029] FIG. 11, panels A1-J5. SA at 45°C reactions under fluorescence microscopy. Polydisperse aqueous microdrops showing the SA annealing fluorescent quenching reactions under a fluorescent microscope. Scale bar = 20pm.

[0030] FIG. 12. Scheme for the TMSD fluorescence assay of modified 56 nt oligonucleotides, F-Tar (-) and Q-Tar (+).

[0031] FIG. 13. Predicted structures of DNA oligos. Putative structures obtained by NuPack1at 25°C and 45°C of all the oligos used in SA and TMSD reactions.

[0032] FIG. 14, panels A1-E2. Chaperoning of the model TMSD reaction with soluble ELP (25°C) and inside a protein-rich condensates upon ELPs phase separation at (45°). TMSD fluorescence assay to observe the real-time displacement of 0.1 pM F-Tar(-)*Q-Tar(+) upon the addition of 0.5 pM 5nt-Tar(+) with and without protein at 25°C (Al-El) and 45°C (A2- E2). Green curves correspond to the positive control of 0.1 pM F-Tar(-)»5nt-Tar(+). Grey curves are negative controls 0. 1 pM F-Tar(-)»Q-Tar(+). Red curves are the TMSD reactions. FIG. 15, panels A1-J5. Strand displacement reactions at 45°C under fluorescence microscopy. Polydisperse aqueous microdrops showing fluorescence unquenching upon SD reactions. Scale bar = 20pm.

[0033] FIG. 16. Experimental workflow for demonstration of chaperoning of NA annealing, subsequent release, and removal of H-24, a switchable SNAC.

[0034] FIG. 17. Results from fluorescence quenching assays to estimate the extent of bimolecular reaction for hybridization of two self-folded complementary oligoDNA strands (Tar(+) and Tar(-), 0.1 pM each) at room temperature in low ionic strength buffers of different pH.

[0035] FIG. 18, panels A1-B3. Extent of bimolecular reaction for hybridization of Tar(+) and Tar(-) (0.1 pM each) at pH 6.5 and 8.5 in the presence of El-80 and E1.40COR30 (room temperature in low ionic strength buffers). Panel A: El-80. Al : 5 pM, A2: 50 pM, A3: 500 pM. Panel B: E1.40COR30. Al: 5 pM, A2: 50 pM. A3: 500 pM.

[0036] FIGS. 19A-19C. Extent of bimolecular reaction for hybridization of Tar(+) and Tar(-) (0.1 pM each) at pH 6.5 and 8.5 in the presence of H-24 (room temperature in low ionic strength buffers). A. 500 nM H-24 B. 5 pM H-24. C. 50 pM H-24.

[0037] FIGS. 20A and 20B. Extent of bimolecular reaction for hybridization of Tar(+) and Tar(-) (0.1 pM each) at pH 6.5 and 8.5 in the presence of 500 nM H-20 (room temperature in low ionic strength buffers). B. Molecular charge on H-24 and H20 as a function of pH as predicted by SnapGene® software (from Dotmatics; available at snapgene.com).

[0038] FIG. 21. Bimolecular hybridization of Tar(+) and Tar(-) (0.1 pM each) at pH 6.5 and 8.5 in the presence of increasing concentrations of H-24 (reaction time: 10 min; room temperature in low ionic strength buffers). 2.5% agarose gels were stained with SyBr Gold.

[0039] FIG. 22. ssDNA and dsDNA localize in the protein-rich phase after LLPS of H-24 at pH 6.5 and in the protein-poor phase after LLPS at pH 8.5.

[0040] FIG. 23. Localization of fluorescently labelled DNA after liquid-liquid phase separation at pH>6.5.

[0041] FIGS. 24A and 24B. DNA and protein gels to assess localization of DNA and SNAC localization after chaperoning of strand annealing from reactants specified in Table 3 (reactions 1,2,3) and subsequent phase separation. A. Agarose gels for visualization of DNA electrophoresis (see text for details). B. SDS PAGE for visualization of presence of protein. PPP: protein-poor phase. PRP: protein-rich phase.

[0042] DETAILED DESCRIPTION OF THE INVENTION

[0043] Reference will now be made in detail to certain aspects of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0044] Nucleic acid hybridization reactions are central to molecular biology and nucleic acid nanotechnology7, utilizing the predictable nature of base-pairing to form intricate structures and dynamic systems. However, these reactions often encounter notable kinetic barriers, particularly when nucleic acids adopt self-folded or constrained conformations, which can reduce the efficiency of hybridization processes. Traditional methods to address these barriers include increasing reactant concentrations or using nucleic acid chaperone proteins, such as viral capsid proteins or proteins with disordered regions. While these approaches can improve hybridization, they often lack precision, scalability, and effective strategies for removing chaperones from reaction products, which is important for subsequent applications in nucleic acid nanotechnology and molecular biology.

[0045] The present disclosure addresses these limitations by introducing smart nucleic acid chaperones (SNACs), which combine the catalytic functions of nucleic acid chaperones with proteins characterized by disordered regions capable of undergoing liquid-liquid phase separation (LLPS). This combination enables programmable, reversible, and environmentally responsive control over nucleic acid hybridization reactions. SNACs concentrate nucleic acids within protein-rich coacervates, significantly accelerating hybridization kinetics-up to 54-fold compared to conventional methods. Additionally, the disclosure incorporates pH-switchable SNACs, allowing precise control over nucleic acid binding and release. This feature facilitates clean separation of assembled nucleic acid structures from the chaperone proteins, addressing a significant gap in current methodologies.

[0046] By utilizing the distinct characteristics of elastin-like polypeptides (ELPs) and their fusion with nucleic acid chaperone proteins, the described platform offers a reliable and adaptable approach for improving nucleic acid hybridization, constructing nanostructures, and separating reaction products. The capability to initiate and reverse LLPS, along with pH- dependent binding, demonstrates a notable progression in nucleic acid technology', facilitating effective, scalable, and refined assembly processes for various applications.

[0047] Definitions

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary' skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, several embodiments with regards to methods and materials are described herein. As used herein, each of the following terms has the meaning associated with it in this section.

[0049] For the purposes of clarity and a concise description, features can be described herein as part of the same or separate embodiments; however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.

[0050] References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

[0051] The phrase “and / or,’" as used herein, should be understood to mean “either or both"’ of the elements so conjoined, e.g., elements that are conjunctively present in some cases and disjunctively present in other cases.

[0052] As used herein, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating a listing of items, “and / or” or “or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one, of a number of items, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only7one of’ or “exactly7one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or"’ as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0053] As used herein, the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof, are intended to be inclusive similar to the term “comprising.” The terms “comprises,” “comprising,” and the like can have the meaning ascribed to them in U.S. Patent Law and can mean “includes,” “including” and the like. As used herein, “including” or “includes” or the like means including, without limitation.

[0054] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1. 1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0055] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.” In addition, it is to be understood that the phraseology7or terminology employed herein, and not otherw ise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section.

[0056] In the methods described herein, the acts can be carried out in a specific order as recited herein. Alternatively, in any aspect(s) disclosed herein, specific acts may be carried out in any order without departing from the principles of the invention, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately or the plain meaning of the claims would require it. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0057] The term '‘about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range and includes the exact stated value or range.

[0058] The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%. or at least about 99.999% or more, or 100%. The term '‘substantially free of’ as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that about 0 wt% to about 5 wt% of the composition is the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

[0059] “Complementary” as used herein refers to the broad concept of subunit sequence complementarity between two nucleic acids, e.g., two DNA molecules. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are complementary to each other when a substantial number (at least 50%) of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T and G:C nucleotide pairs). Thus, it is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing’’) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand w hich is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. For example, in some aspects, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, including at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In some aspects, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

[0060] As used herein, the term ‘'hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the length of the formed hybrid, and the G:C ratio within the nucleic acids. Nucleic acid hybridization is the process where single-stranded nucleic acid molecules (DNA or RNA) anneal or bind to complementary sequences by forming hydrogen bonds between their base pairs, creating double-stranded hybrids. This process depends on the sequence complementarity between the strands, usually following Watson-Crick base pairing rules (A pairs with T or U, and G pairs with C). Hybridization can occur in various forms: DNA-DNA, DNA-RNA, or RNA-RNA interactions.

[0061] The term “nucleic acid” typically refers to large polynucleotides. By “nucleic acid” is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

[0062] As used herein, the term “nucleic acid” encompasses RNA as well as single and double stranded DNA and cDNA. Furthermore, the terms, “nucleic acid,” “DNA,” "RNA” and similar terms also include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, the so called “peptide nucleic acids,” which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. By “nucleic acid” is meant any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate. phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil). Conventional notation is used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5 ’-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5 ’-direction. The direction of 5’ to 3’ addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the “coding strand’’; sequences on the DNA strand which are located 5’ to a reference point on the DNA are referred to as “upstream sequences”; sequences on the DNA strand which are 3’ to a reference point on the DNA are referred to as "downstream sequences.” Nucleic acids can comprise hairpin structures (nucleic acid hairpin, also known as a stem-loop structure, is a secondary structure in nucleic acid molecules that forms when two regions of the same strand base-pair to form a double helix that ends in an unpaired loop).

[0063] The term “nucleic acid construct,” as used herein, encompasses DNA and RNA sequences encoding the particular gene or gene fragment desired, whether obtained by genomic or synthetic methods.

[0064] “Homologous” or “identity” as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in tw o compound sequences are homologous then the tw o sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology. By way of example, the DNA sequences 3'ATTGCC5' and 3'TATGGC share 50% homology. As used herein, “homology ” is used synonymously with “identity.”

[0065] The determination of percent identity between two nucleotide sequences can be accomplished using a mathematical algorithm. For example, a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990, J. Mol. Biol. 215:403-410), and can be accessed, for example at the National Center for Biotechnology' Information (NCBI) world wide web site. BLAST nucleotide searches can be performed with the NBLAST program (designated “blastn” at the NCBI web site), using the following parameters: gap penalty' = 5; gap extension penalty' = 2; mismatch penalty = 3; match reward = 1; expectation value 10.0; and word size = 11 to obtain nucleotide sequences homologous to a nucleic acid described herein. BLAST protein searches can be performed with the XBLAST program (designated “blastn” at the NCBI web site) or the NCBI “blastp” program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes. Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402). Alternatively, PSLBlast or PHLBlast can be used to perform an iterated search which detects distant relationships between molecules (Id.) and relationships between molecules which share a common pattern. When utilizing BLAST. Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs (e g., XBLAST and NBLAST) can be used.

[0066] The percent identity' between tw o sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, ty pically exact matches are counted.

[0067] In some aspects, the nucleic acids are isolated from or included in a sample, such as a physiologically’ or environmentally related samples, including, but not limited to, body fluids or body tissues, such as blood, serum, plasma, aspirate, stool, urine, mucus, saliva, sputum, nasopharyngeal discharge, cells, tissue or a combination thereof.

[0068] Smart nucleic acid chaperone (SNAC) refers to proteins or molecules that help nucleic acids achieve desired conformations or aid in gene regulation. Intrinsically disordered protein (IDP) refers to a protein or protein region that does not have a fixed, stable three-dimensional structure under physiological conditions, either in whole or in part. Instead, IDPs exist as flexible, dynamic conformational ensembles that fluctuate between multiple structural states. This lack of fixed structure is encoded in their amino acid sequence, which generally features a high proportion of charged and polar residues and a low proportion of hydrophobic residues, preventing the formation of stable folded domains.

[0069] IDPs and intrinsically disordered regions (IDRs) are highly prevalent in biology and play roles in cellular processes such as signaling, regulation, cell division, DNA replication and repair, and response to external stimuli. Their flexibility enables them to interact with multiple different partners, often facilitating complex regulatory and signaling networks. Some IDPs or IDRs may adopt more ordered structures when binding to specific molecular partners, allowing for functional versatility.

[0070] Elastin-like polypeptide (ELP) refers to a genetically engineered protein-based biopoly mer inspired by human tropoelastin, the precursor to natural elastin found in mammals. ELPs are characterized by a repeating amino acid sequence, typically (VPGXG) / ? (SEQ ID NO; 1), where "V” is valine, “P’?is proline, ”G is glycine, "X can be any amino acid except proline, and ‘"n” denotes the number of pentapeptide repeats. The choice of “X” and the length of the repeat allow one to tune the properties of ELPs, such as their temperature responsiveness. ELPs can exhibit a unique lower critical solution temperature (LOST) phase transition, meaning they are soluble at lower temperatures but aggregate when the temperature surpasses a specific threshold (the transition temperature, Tt). Above this temperature, ELPs coalesce into a reversible, amorphous phase known as a coacervate. ELPs are intrinsically disordered below' the transition temperature, giving them significant flexibility and dynamic structural properties similar to natural elastin. Their stimuli-responsiveness, biocompatibility, and tunable material properties make ELPs valuable for applications in drug delivery, biomaterials, nanotechnology, and tissue engineering. ELPs are synthesized through genetic engineering, where their DNA sequences are designed and expressed in host organisms to produce the desired polypeptide chain. Thus, ELPs are customizable, reversible, thermoresponsive biopolymers modeled on elastin, widely utilized in biomedical and materials science for their disordered structure and unique phase behavior. Fusion proteins or chimeric proteins are proteins created through the joining of two or more genes or portions thereof (so that they are transcribed and translated as a single unit, producing a single polypeptide) that originally coded for separate proteins or portions thereof.

[0071] As described herein, polymers suitable for use in the methods can comprise proteins or synthetic polymers that exhibit temperature triggered phase separation and that incorporate pH switchable ionizable groups. For example, the synthetic polymer can be a poly(JV-isopropyl acrylamide) (PNIPAAm). An example of pH switchable ionizable groups that could be incorporated synthetically into PNIPAAm are imidazole groups such as the side chain of histidine. Other polymers include, but are not limited to, poly(diallyldimethylammonium chloride) (PDADMAC) with poly (styrenesulfonate) (PSS), poly(allylamine hydrochloride) (PAH) with PSS (anion); poly(ethyleneimine) (PEI) with poly(acrvlic acid) (PAA); PDMAEMA-based cationic blocks (e.g., PEG-b-PDMAEMA) with anionic partners such as poly(acrylic acid) or poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS); PAA with quaternary ammonium polymers such as poly(vinyl benzyl trimethylammonium chloride); PAMPS with cationic blocks like PEG-b-PDMAEMA or with other amine-containing polymers; PEG-b-PAA or PEG-b-PAMPS with complementary cationic polypeptides or synthetic polycations to give polymer-polymer or polymer-peptide coacervates; block copolymers combining PNIPAM with a charged block (e.g., PNIPAM-b-PSS) that yield micellar or droplet-like coacervate phases in water or a combination thereof.

[0072] In another example, the polymer can be an intrinsically disordered protein (IDP) comprising an amino acid composition that exhibits temperature triggered phase separation. Exemplary IDPs include poly cationic ELPs, collagen, elastins, resilins, RRM-RGG and HCV Core proteins, and polypeptides comprising amino acid repeats rich in proline and glycine. The polypeptides can be modified or "tuned" to exhibit soluble to insoluble phase transitions that are of interest, including a lower critical solution temperature (LCST) transition that occurs upon heating above a critical solution temperature or an upper critical solution temperature (UCST) transition that occurs upon cooling below7a critical temperature. See Quiroz, F., Chilkoti, A., Sequence heuristics to encode phase behavior in intrinsically disordered protein polymers, Ant. Mater. (2015); 14(11): 1164, which is incorporated herein by reference.

[0073] Liquid-liquid phase separation (LLPS) refers to a biophysical process where biomolecules, primarily proteins and nucleic acids, spontaneously segregate into two distinct liquid phases, forming condensed, dynamic, membrane-less compartments w ithin cells called biomolecular condensates or membraneless organelles. This phenomenon is similar to the separation of oil and water and is driven by multivalent interactions among molecules, often involving intrinsically disordered regions (IDRs) of proteins. LLPS enables the formation of cellular compartments without membranes, such as nucleoli, P granules, stress granules, and other nuclear or cytoplasmic bodies, allowing cells to spatially organize biochemical reactions, regulate molecular interactions, and rapidly respond to environmental changes. These condensates are dynamic and reversible, contributing to various biological processes including gene regulation, DNA repair, and signal transduction.

[0074] Protein-rich coacervates refers to condensed, liquid-like phases formed via liquid-liquid phase separation (LLPS), characterized by a high concentration of proteins and often other biomolecules such as nucleic acids. These coacervates arise chiefly through weak, multivalent, and often electrostatic interactions among proteins-particularly those with intrinsically disordered regions (IDRs)-sometimes assisted by other charged biopolymers, resulting in a dense phase that coexists with a dilute surrounding phase. The protein composition in these coacervates tends to be complex, and their structure is typically amorphous and dynamic rather than rigid or crystalline. The formation and stability of protein-rich coacervates depend on factors including protein charge, sequence complexity, hydrophobicity, and presence of multiple interacting motifs or peptides. Protein coacervates serve as models for membraneless organelles in cells, providing microenvironments that facilitate biochemical reactions and biological organization.

[0075] RNA recognition motif (RRM) refers to a highly conserved RNA-binding protein domain, one of the most abundant and versatile motifs in eukaryotic proteins involved in RNA metabolism. The typical RRM domain consists of about 80-100 amino acids and has a characteristic fold composed of a four-stranded antiparallel 0-sheet packed against two a- helices, forming a |3-a-|3-(>-a-(> structural topology. Key structural features include two highly conserv ed sequence motifs, RNP1 and RNP2, located on the central 0-strands, which mediate specific binding primarily to single-stranded RNA sequences, although some RRMs can also bind DNA or proteins. The -sheet surface usually serves as the primary RNA interaction platform, with side chains stacking with RNA bases to confer affinity and sequence specificity. RRMs functionally participate in diverse RNA-related processes such as splicing, stability, export, and translation by recognizing short RNA motifs or structured RNA elements, often cooperating with other RRMs to enhance binding specificity and affinity. The domain is adaptable, with variations in loop regions and secondary structures allowing recognition of different RNA sequences or structures. Examples of proteins containing RNA Recognition Motif (RRM) domains include, but are not limited to, heterogeneous nuclear ribonucleoproteins (hnRNPs), such as hnRNP Al, hnRNP A2 / B1, and hnRNP C 1 / C2; embryonic lethal abnormal vision (ELAV) family proteins; spliceosomal proteins like U1A, U1 70k, and U2B"; polypyrimidine tract-binding protein (PTB); Poly(A)-binding proteins (PABPs) and / or TDP-43 and FUS, RNA-binding proteins linked to neurodegenerative diseases and containing RRMs with diverse RNA recognition specificities. The human FUS (Fused in Sarcoma) protein consists of 526 amino acids. Its sequence begins with a serine-, tyrosine-, glycine-, and glutamine-rich (SYGQ-rich; SEQ ID NO: 2) N-terminal region and includes domains such as an RNA recognition motif (RRM), multiple arginine-glycine-glycine (RGG) repeat regions, a C2C2 zinc finger motif, and a nuclear localization signal (NLS) at the C- terminus.

[0076] Arginine-glycine (RGG), also known as an RGG box, is a protein domain characterized by multiple repeats of the arginine-glycine-glycine (RGG) or arginine-glycine (RG) sequence motifs clustered within a stretch of about 30 ammo acids. These domains are intrinsically disordered, meaning they lack a fixed three-dimensional structure in isolation, which allows them conformational flexibility and adaptability to interact with various RNA molecules and proteins. The positively charged arginine residues mediate electrostatic interactions with the negatively charged nucleic acid backbone, while the glycine residues confer flexibility. RGG domains can act synergistically with other RNA / DNA-binding domains like RNA Recognition Motifs (RRMs) to regulate RNA metabolism, including transcription, splicing, translation, and regulation of RNA stability. Additionally, RGG domains contribute to the formation of protein- RNA condensates through liquid-liquid phase separation, playing roles in cellular organization and stress response. Proteins such as FUS, nucleolin, and hnRNP Al contain RGG domains used for their RNA and / or DNA-binding functions and biological activities.

[0077] Hepatitis C virus core protein (HCV CP) refers to a structural protein that forms the viral nucleocapsid by surrounding and protecting the viral RNA genome. It is initially part of a large polyprotein that is cleaved by host and viral proteases to yield the mature core protein. The mature HCV core protein is released into the cytoplasm and localizes primarily to lipid droplets and endoplasmic reticulum membranes, playing a role in viral particle assembly and release. Structurally, the HCV core protein is composed of three domains: domain 1 (DI), located at the N-terminus, is rich in basic amino acids and contains RNA-binding clusters essential for binding the viral genomic RNA; Domain 2 (D2) includes two alpha-helical modules and a hydrophobic region crucial for membrane binding and stable folding; it interfaces with lipid droplets and host membranes; and Domain 3 (D3), at the C-terminus, is highly hydrophobic and acts as a signal sequence for the viral envelope glycoprotein El; this transmembrane region is cleaved off to form the mature core protein. Functionally, HCV core serves as an RNA chaperone, facilitating proper folding and strand annealing of viral RNA to ensure efficient replication and genome packaging. It also interacts with various host factors to regulate viral assembly and release.

[0078] Toehold-mediated strand displacement (TMSD) refers to an enzyme-free nucleic acid reaction mechanism where a single-stranded "invading" DNA or RNA strand binds to a short single-stranded overhang region (called the "toehold") on a double-stranded nucleic acid complex. This initial binding to the toehold initiates a branch migration process in which the invading strand progressively displaces one of the original strands (the incumbent strand) by forming base pairs with the complementary strand, ultimately replacing the incumbent. The toehold serves as a foothold that greatly enhances the reaction speci licity and kinetics, allowing the displacement to proceed efficiently and directionally. The reaction is energetically favorable and highly programmable, with the toehold length and sequence determining the rate and selectivity of strand displacement. This mechanism is widely used in DNA nanotechnology, molecular computation, and biosensing to control DNA or RNA hybridization reactions without enzymes.

[0079] Transition temperature (Tt) refers to the characteristic temperature at which an intrinsically disordered protein polymer such as an elastin-like polypeptide (ELP) undergoes a reversible lower critical solution temperature (LCST) phase transition in aqueous solution. Below Tt, the ELP is soluble and dispersed; at or above Tt, the ELP demixes from the surrounding solution via liquid-liquid phase separation (LLPS) to form a concentrated, proteinrich coacervate phase that coexists with a dilute, protein-poor phase. Tt is sequence- and environment-dependent and can be modulated by factors including the ELP guest residue (X in VPGXG; SEQ ID NO: 1), polymer length, concentration, ionic strength, pH, cosolutes (e.g., salts, crowding agents), and the presence of nucleic acids that may lower Tt by promoting condensate formation. In practice, Tt is determined by monitoring solution turbidity or absorbance as a function of temperature and identifying the inflection point (e g., the maximum in the first derivative of absorbance versus temperature) corresponding to onset of phase separation. pH-switchable smart nucleic acid chaperone (SNAC) refers to a nucleic acid-binding protein construct whose affinity for nucleic acids and propensity to undergo liquid-liquid phase separation (LLPS) can be reversibly toggled by changing solution pH. A pH-switchable SNAC typically comprises an intrinsically disordered protein (IDP) domain, such as an elastin-like polypeptide (ELP) enriched in titratable residues (e.g., histidines), optionally fused to a nucleic acid chaperone module (e.g., RRM and / or RGG domain or a viral capsid fragment). At lower pH (e.g., about pH 6.0 to about 7.0, including about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9 or about 7.0), protonation of titratable side chains increases net positive charge, promoting electrostatic attraction to the negatively charged nucleic acid backbone and favoring LLPS into protein-rich coacervates that recruit and concentrate nucleic acids. At higher pH (e.g., about 8.0 to about 9.0, including about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9 or about 9.0), deprotonation reduces or reverses net charge, diminishing nucleic acid binding and dissolving or releasing nucleic acids from coacervates, thereby enabling clean separation of assembled nucleic acid products from the SNAC. The switching behavior can be exploited to

[0080] (i) accelerate hybridization or assembly within condensates under binding pH conditions and

[0081] (ii) subsequently release and isolate products under non-binding pH conditions.

[0082] Self-complementary regions refers to sequences within a single strand of DNA or RNA that are complementary to other regions of the same strand, allowing it to fold back on itself and form double-stranded structures by base pairing. These regions enable the formation of secondary structures such as hairpin loops, stem-loops, bulges, or internal loops where the sequence pairs with its own complementary segment through hydrogen bonds, typically following the Watson-Crick base pairing rules (A with T or U, and G with C). This folding is used in the structural and functional diversity of RNA molecules and can also occur in singlestranded DNA sequences. Self-complementarity is important in many biological processes and molecular biology techniques, as it can influence the stability, folding, and interactions of nucleic acids. For example, hairpin loops formed by self-complementary sequences are often involved in regulation of gene expression and RNA processing.

[0083] Methods involving conventional molecular biology' techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises, such as Molecular Cloning: A Laboratory' Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Carruthers, Tetra. Letts. 22: 1859-1862, 1981, and Matteucci et al., J. Am. Chem. Soc. 103:3185. 1981.

[0084] NA Chaperones. ELPs and IDPs

[0085] NA Chaperones

[0086] While nucleic acid (NA) chaperones and phase-separating proteins have been studied independently, there remains a need for “smart” systems that combine these functions to enable enhanced control over NA hybridization reactions. Additionally, current methods lack effective ways to remove chaperone proteins from reaction products after assembly is complete. The ability to trigger assembly and then cleanly separate the assembled NA structures from the chaperone proteins would enable new applications in NA nanotechnology and molecular biology.

[0087] Nucleic acid chaperones are a class of proteins that facilitate formation of natively folded RNAs and DNAs. These chaperones are involved in many nucleic acid-dependent processes. Examples of nucleic acid chaperones include the retroviral nucleocapsid (NC) proteins and Gag polyproteins, nucleocapsid protein of the hepatitis C virus, human immunodeficiency virus type-1 (HIV-1) nucleocapsid protein (NC), also those from retrotransposons LINE-1 and Ty3, ORF Ip and Ty3 NC, Human hepatitis B virus core protein (the arginine-rich domain of this protein has nucleic acid chaperone activity'), arginine-glycine (RGG) domains, and artificial or recombinant chaperones. Other nucleic acid chaperones include, but are not limited to, Hfq (Helicases from the Fis family), cold shock proteins (CSPs), heat shock proteins (HSPs), ribosomal proteins, including SI and S 12, the Sm family of proteins, FinO / ProQ family proteins, and RNA binding proteins.

[0088] ELPs / IDPs

[0089] Elastin-like polypeptides (ELPs) are synthetic biopolymers with applications in the fields of cancer therapy, tissue scaffolding, metal recovery, and protein purification. The ability of ELPs to undergo morphological changes at certain temperatures enables specific proteins or other molecules that are bound to the ELPs to be separated out from the rest of the solution via experimental techniques such as centrifugation. The general structure of polymeric ELPs is (VPGXG)n (SEQ ID NO: 1), where the monomeric unit is Val-Pro-Gly-X-Gly (SEQID NO: 1), and the "X" denotes a variable amino acid that can have consequences on the general properties of the ELP, such as the transition temperature (Tt). Specifically, the hydrophilicity or hydrophobicity and the presence or absence of a charge on the variable residue play a role in determining the Tt. Also, the solubilization of the variable residue can affect the Tt. The "n" denotes the number of monomeric units that comprise the polymer. In general, these polymers are linear below the Tt but aggregate into spherical clumps above the Tt.

[0090] Although engineered and modified in a laboratory setting, ELPs share structural characteristics with intrinsically disordered proteins (IDPs) naturally found in the body, such as tropoelastin. The repeat sequences found in the biopolymer give each ELP a distinct structure, as well as influence the lower critical solution temperature (LCST), also referred to commonly as the Tt. It is at this temperature that the ELPs move from a linear, relatively disordered state to a more densely aggregated, partially ordered state. Although given as a single temperature, Tt, the ELP phase change process generally begins and ends within a temperature range of approximately 2 °C. Also, Tt is altered by the addition of unique proteins to the free ELPs.

[0091] The transition temperature of an ELP can depend to a certain extent on the identity of the "X" residue found at the fourth position of the pentapeptide monomeric unit. Residues that are highly hydrophobic, such as leucine and phenylalanine, tend to decrease the transition temperature. On the other hand, residues that are highly hydrophilic, such as serine and glutamine, tend to increase the transition temperature. The presence of a potentially charged residue at the "X" position will determine how the ELP responds to varying pHs, with glutamic acid and aspartic acid raising the Tt at pH values in which the residues are deprotonated and lysine and arginine raising the Tt at pH values in which the residues are protonated. The pH needs to be compatible with the charged states of these amino acids in order to raise the Tt. Also, higher molecular mass ELPs and higher concentrations of ELPs in solution make it much easier for the polymer to form aggregates, in effect lowering the experimental Tt.

[0092] Oftentimes, ELPs are not used in isolation, but are rather fused with other proteins to become functionally active, such as provided herein generating a fusion protein with an ELP and a NA chaperone protein. The structure of these other proteins will have a certain effect on transition temperature.

[0093] ELPs are protein-based biopolymers and synthesis involves can involve synthetic processes or manipulation of genes to continually express the monomeric repeat unit. Various techniques have been employed in the production of ELPs of various sizes, including unidirectional ligation or concatemerization. overlap extension polymerase chain reaction (OEPCR), and recursive directional ligation (RDL).

[0094] Due to the unique temperature-dependent phase transition experienced by ELPs, in which they move from a linear state to a spherical aggregate state above their Tt, as well as the ability of ELPs to be easily conjugated with other compounds, these biopolymers hold numerous applications. Some of these applications involve ELP use in protein purification, cancer therapy, and tissue scaffolding.

[0095] EXAMPLES

[0096] Various aspects of the present invention can be better understood by reference to the following Examples which are offered by way of illustration. The present invention is not limited to the Examples given herein.

[0097] Summary

[0098] Nucleic acid (NA) hybridization reactions are of central importance in molecular biology as well as in methods associated with NA nanotechnologies, which take advantage of the programmable and predictable nature of base-pairing to generate intricate structures and dynamic, responsive reaction systems. NA chaperones are molecules (typically proteins) that can catalyze the formation of thermodynamically favorable NA structures through inter- and intramolecular hybridization. Provided herein are smart NA chaperones (SNACs) which combine the catalytic functions of NA chaperones with protein polymers that undergo programmable, triggered and reversible liquid-liquid phase separation (LLPS). The ‘'smart” nomenclature used harkens back to “smart polymers”, a general term for environmentally sensitive polymers that have been explored for myriad technological applications. Protein polymers that undergo reversible LLPS are intrinsically disordered proteins (IDPs) or proteins with significant intrinsically disordered regions in their amino acid sequence. Thus, presented herein are SNACs that are fusions of known NA chaperones and IDPs or are IDPs that have been designed to have NA chaperoning function. This work demonstrates for the first time the enhanced NA chaperoning function of SNACs in phase-separated polymer rich coacervates and switchable SNACs in which chaperoning can be followed by a convenient molecular separation step to remove the SNAC from the desired, assembled NA architecture. Oligonucleotide strand annealing and toehold mediated strand displacement are used as model reactions to demonstrate the function of SNACs and their power as nano-assemblers.

[0099] Introduction

[0100] Nucleic acids (NAs) are molecules that carry the genetic information of all forms of life that dictates the function of an organism’s cells, tissues and organs. Reactions involving NAs are central to many biological processes in vitro and in vivo, such as transcription, translation, apoptosis, stress responses. Often, these NA processes are influenced by DNA and RNA binding proteins (DRBPs).1In biology, the proteins that assist in the structural changes of macromolecules and therefore may influence in the kinetics of molecular reactions are called chaperones. DNA binding proteins are associated with regulating genomic functions such as replication, transcription, or repair via specific or promiscuous interactions.2RNA binding proteins are known to regulate gene expression via regulation of RNA paths and function.3An example is the stress-induced formation of ribonucleoprotein (RNP) membrane-less organelles (MLOs) to regulate mRNA translation and localization.4Among DRBPs, it is accepted that intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) of proteins can play a significant role in N A-binding.2 4IDPs (and IDRs) are proteins (and protein regions) that lack a well-defined three-dimensional structure, breaking the paradigm of structure-protein function as IDPs are involved in crucial biological processes like cell signaling, MLO formation or viral packaging5,6Provided herein is the exploration of the interactions of IDPs and DNA and their influence on DNA hybridization reactions using the biosynthetic, genetically engineered family of IDPs named elastin-like-polypeptides (ELPs). ELPs are synthetic IDPs that may be used to engineer MLOs due to their spontaneous and reversible low critical solution temperature (LCST) behavior in water. They can be expressed in large quantities in standard E. coli bioreactor systems.7ELPs consist of repetitive, low complexity sequences of V-P-G-X-G (SEQ ID NO: 1) (X can be any amino acid, except proline) that phase separate upon thermal stimulus to undergo liquid-liquid phase separation (LLPS) induced simple coacervation to form protein-rich condensates.8ELPs and their fusions were selected as model NA binding and chaperoning constructs because they can be conveniently fused to other proteins at the genetic level to adopt new and robust NA binding functionalities while maintaining their LCST behavior.9 12Moreover, the phase behavior of ELPs has been characterized and is easily tunable. Also. ELPs and ELP fusions have been previously explored as NA-binding proteins in solution and upon coacervate formation forming DNA and RNA rich MLOs.13

[0101] Provided herein are several approaches to engineer NA-binding ELPs and ELP-fusions to explore their dual capacity for binding DNA (either in soluble nucleoprotein (NP) complexes or as phase-separated protein-DNA condensates, i.e., complex coacervates) and for their use as molecular chaperones of DNA reactions. The term smart NA chaperones (SNACs) is coined to denote NA chaperones that also have the function of triggerable phase separation (coacervation). The modifier smart comes from the literature on environmentally sensitive polymers which can change their properties in response to small changes in environmental stimuli and thus have been widely investigated for numerous technological applications. Herein is described the development of SNACs for annealing and displacement reactions of DNA or RNA which are at the heart of molecular biology, NA nanotechnology, and molecular logic devices.

[0102] EXAMPLE I

[0103] One phenomena occurring between NAs is strand annealing, the process in which single-stranded (ss) DNA and / or RNA molecules complex to their complementary DNA and RNA forming double-stranded intramolecular and supramolecular assemblies.14DNA and RNA annealing is essential in cellular processes such as transcription, translation, replication or DNA repair,15and in molecular biology laboratory techniques such as NA amplification (e.g., PCR, LAMP, NASBA), and DNA sequencing.16Often, these reactions have significant kinetic barriers that can be overcome by increasing reactant concentrations or through the use of protein chaperones.17 19Mechanisms of protein chaperoning of complementary DNA hybridization reactions can be related to NA structural changes via folding or misfolding into thermodynamically stable and available structures,19or electrostatic interactions that bring together complementary sequences.20Interestingly, IDPs, such as StpA or FinO, are NA chaperones due to their structural flexibility7and dynamics.21,22Moreover, other NA chaperone proteins include viral capsid proteins,23such as the hepatitis C virus core protein (HCV CP), HIV-1 nucleocapsid protein (NC), or the dengue virus 2 capsid (DENV2C), which were known initially to chaperone packaging of viral genomes,24but that have been found useful in chaperoning of DNA strand annealing and displacement reactions.23 29 Three different NA-binding ELPs (Table 1) were engineered to explore their chaperoning activity in DNA strand annealing (SA) reactions in their soluble and phase- separated states. First, previously studied ELP E3 was used. ELP E3 is a positively charged ELP (Table 1) with no binding activity in solution below the Tt (Figure 6), but that can model a DNA-rich MLO by recruiting ssDNA into its coacervate upon LLPS.13Next, an ELP named E1-40.COR30 was engineered. E1-40.COR30 is comprised of forty canonical, VPGVG (SEQ ID NO: 3), pentameric repeats (El -40), with no inherent NA binding activity7fused to 30 aa (COR30) from the HCV CP (Table 1), a known DNA chaperone.1731131With this approach, the DNA binding activity from the COR30 region and the phase behavior from the El -40 region will be conferred to the engineered fusion construct. Finally, a third ELP named E3.10 was engineered. E3.10 is the fusion of two, well-known NA-binding and chaperone domains from FUS proteins, the RNA recognition motif (RRM) and the arginine-glycine 2 (RGG) domain (Table I)32With this design, the objective is to maintain the LLPS of E3 along with its weak DNA binding behavior (above Tt) and improve the NA binding capacity by adding RRM and RGG. Chaperoning of DNA annealing and displacement reactions has been widely studied, but the behavior of these reactions in MLO-like organelles is yet to be explored. Here it is shown that E1-40.COR30 and E3.10 are potent DNA chaperones that can overcome the kinetic barrier to anneal highly structured and self-complementary' ssDNA oligos (Tar (+) and Tar (-), Table 3) in their soluble state. Moreover, above their LCST, all three ELPs form concentrated protein- DNA coacervates that can speed up the kinetics of hybridization reactions such as those between Tar(+) and Tar(-) DNA.

[0104] Table 1: ELP sequences for the NA binding proteins studied herein. XI represents the ELP guest residue in the E3 construct. Exemplary sequences for these proteins are given in Tables 2 and 2.1. Materials and Methods.

[0105] Materials.

[0106] Expression vector pET24 was purchased from Novagen, Inc (Milwaukee, WI); One- Shot BL21 Star(DE3) Escherichia coli cells were from ThermoFisher Scientific (Waltham, MA); restriction enzymes were from New England Biolabs (Beverly, MA). DNA purification kits were purchased from QIAGEN. Inc. (Valencia, CA). DNA sequences (genes and oligos) were purchased from Integrated DNA Technologies (Coralville, IA). Luria Broth (LB) agar plates were purchased from Bacto Agar, Becton Dickinson (Franklin Lakes, NJ), and Millipore Sigma (St. Louis, MO); kanamycin was from Ultrapure, VWR, (Radnor, PA); LB Broth and Terrific Broth (TB) from IBI Scientific (Dubuque, Iowa).

[0107] Gene synthesis.

[0108] The plasmid containing the sequence for E3 was synthesized in a previous study using recursive directional ligation.12,30Plasmids that encode El-40. COR30 and E3.10 were constructed using recursive directional ligation.33In brief, ELP sequences are created byannealing complementary ssDNA sequences encoding the desired amino acid composition and cloning them into a PET-24+ vector. First, an ELP monomer is engineered into the vector. Then, two different pairs of restriction enzymes digest the vector to create two complementary cut vector populations. Next, the two digested vectors are ligated together, doubling the monomer sequence. The process is repeated until the desired length of protein polymer is reached. This method was also used to introduce other protein sequences that were not ELPs, such as COR30, RRM, and RGG. Later, the genes for the protein sequences were transformed into chemically competent E. coli cells (BL21).

[0109] Protein expression and purification.

[0110] Escherichia coli cells (BL21) encoding the proteins of interest were inoculated into an LB agar plate containing 45 pg / mL kanamycin sulfate. The plate was incubated overnight at 37°C, and bacterial colonies were inoculated the next day into 3 mL of LB broth with 45 pg / mL kanamycin sulfate. This culture was incubated overnight at 220 rpm and 37°C. The culture was then transferred into IL of TB with 45 pg / mL kanamycin sulfate, and cells were grown for 6 hr at 220 rpm and 37°C. After 6 hrs, IPTG was added, and cells were incubated at 220 rpm and 37°C for 18 hrs. The culture was then harvested by centrifugation at 4°C and 3000 rpm for 30 min. Pellets were then resuspended into a lysis buffer (lx phosphate buffered saline (PBS), 1 protease inhibitor tablet, 50 mL, 0.5M, and pH 8.0 ethylenediaminetetraacetic acid (EDTA)). The cell suspension was lysed by sonication to release all intracellular content.

[0111] Proteins were purified by inverse transition cycling, exploiting the reversible thermally responsive protein phase separation inherent to ELP constructs.34The approach consists of cyclic centrifugation steps that alternate between cold (4°C) and hot (40°C) centrifugation in IxPBS until all contaminants are removed. The solution is clear, typically after 2 to 5 rounds. However, for E3. 10, hot centrifugation was replaced by room temperature centrifugation, and LLPS was triggered by adding IM ammonium sulfate instead of heating. Addition of ammonium sulfate helps bring down the required temperature for ELP aggregation from 37° C to 25° C, avoiding possible denaturation of folded domains in the protein.35

[0112] Measurement of NA-ELP binding by gel retardation assay.

[0113] 0.1 pM Tar(+) ssDNA, 0.2 pM Tar(+) • Tar(-) dsDNA, and serial dilutions of El- 40. COR30 or E3. 10 were prepared and incubated at room temperature for 30 min. The sample was then loaded into standard 2.5% agarose gels and run in lx Tris / Borate / EDTA (TBE) buffer at 75V for 60 min at 4°C. The gel is post-stained with SyBr Gold and imaged in a transilluminator.

[0114] UV-VIS absorption spectrophotometry.

[0115] Samples containing E1-40.COR30 or E3.10 and 0.1 pM Tar(+) ssDNA were prepared. To obtain the transition temperature (Tt) for LLPS, absorbance at 380nm as a function of the temperature of the samples was obtained in a temperature-controlled (Peltier temperature controller, Agilent, Santa Clara, CA) UV-vis spectrophotometer (Cary 300 UV-vis, Agilent) as previously described36The Tt is obtained by taking the maximum in the first derivative of the absorbance as a function of temperature. Results in Figure 4B are from optimal ELP concentrations that provide DNA binding and Tt below 45°C needed for all the experiments carried out at 45°C.

[0116] Strand annealing assays via agarose gels.

[0117] Tar(+) and Tar(-) were diluted from IpM stock solution (IxTris / EDTA (TE), pH 7.5). 20pL annealing solutions containing equimolar 0.1 pM concentrations of DNA were prepared with and without protein, as tabulated in Table 4 in sodium phosphate buffer (100 mM). Reactions were incubated at different temperatures for 10 min.17Reactions (and controls) are described in the figure captions. Reactions were stopped by adding 10 pL of a solution containing 20% glycerol, 20 mM EDTA pH 8.0, 0.2% SDS, 0.25 % bromophenol blue.37This solution denatures the protein and releases it from the DNA oligos.37Extent of reactions was resolved in 2.5% agarose gels in IX TBE at 4°C for 60 min at 75V. The gel is post-stained with SyBr Gold and imaged in a transilluminator.

[0118] Table 4: Samples for SA. Detailed sample composition for fluorimetry measurements anc microscopy of SA reactions.

[0119] Strand annealing assays via fluorescence quenching.

[0120] Tar (-) was modified on the 5’ end, substituting the last 5 nt, GGGTT by TAAAG and attaching a fluorophore 3ATTO488 at the 3’ end (IDT, Coralville, IA). With this modification, the newly synthesized 56nt oligo, denoted as F-Tar(-), is complementary to Tar (+) on 51 / 56 nt, leaving a free 5 nucleotide (nt) toehold on both F-Tar (-) and Tar (+) strands upon complementary strand hybridization. The Tar (+) strand was labeled with a 5’ Iowa Black FQ quencher (5IABkFQ), and denoted as Q-Tar(+) (Figure SI, Table S2). 20 pL solutions with equimolar DNA concentrations with and without protein were prepared in triplicate, as noted in Table 4 in sodium phosphate buffer (100 mM) and loaded into a 384-well microtiter plate. Annealing was characterized by a fluorescence decay caused by quenching of ATTO488 by the proximity of 5IABkFQ. For 4 hours, every 2 minutes, samples were excited at 488nm, and fluorescence intensity was measured at 520 nm at 25°C and 45°C. It was assumed that the fluorescence intensity of the reactions was caused by the free fluorescently labeled F-Tar (-), and by comparing it to the negative control, the amount of free DNA was calculated in the solution. The extent of the annealing reaction was calculated using min-max normalization (see equation below) to the fluorescence intensity of a negative control containing only ATTO488- F-Tar(-) (minimum) and a positive control containing only 5IABkFQ-Q-Tar(+) (maximum), with and without protein, at the same fluorescent oligo concentration as the reactions. That is, (Y-F)

[0121] T = where Y’ is the extent of the annealing reaction, Y is the measured fluorescence intensity for the SA reaction, F is the fluorescence measured for F-Tar(-), and Q is that measured for Q-Tar(+). Results were plotted and fitted using GraphPad Prism version 9.3.1 (GraphPad Software, San Diego, CA).

[0122] The fit of a one-phase association model in GraphPad Prism (version 9.3.1, GraphPad Software, San Diego, CA) was used to analyze the kinetics of the annealing reaction. This model describes pseudo-first-order kinetics, typically used for ligand-receptor or enzymesubstrate interactions, where a fraction of the available binding sites becomes occupied over time. The fluorescence intensity data were fit to the following equation: Y = Yo+ Plateau — Fo) X (1 — e^~Kxx^) where Yois the initial fluorescence, "Plateau" represents the maximum fluorescence intensity, K is the rate constant, and x is time. From this fit, the reaction half-time tq^was calculated using the formula tt / 2= ln(2) / K which represents the time required to complete half of the reaction. The fold change in kinetics was determined by comparing the half-times of the protein-containing reactions to the control (no protein). This comparison provided a direct measure of how much the reaction was accelerated in the presence of LLPS. The raw data are presented in Figure 7.

[0123] Brightficld and fluorescence microscopy.

[0124] To image the process of ELP coacervation, F-Tar(-)-ELP interactions, and the progression of the DNA SA reactions in one and two-phase reaction systems as described above, an Olympus 1X83 fluorescence microscope (Olympus Life Science Technology Division, Center Valley, PA) equipped with a heating stage (TS4-MP / ER / PTU, Clifton, NJ) was used. For every sample, polydisperse microdroplets were prepared by mixing an aqueous phase and an oil phase in a microcentrifuge tube and brief manual shaking. First, one droplet was imaged using brightfield and fluorescence acquisition modes to track the temperature- mediated coacervation, DNA capture, and resolubilization of the ELPs in the presence of F- Tar(-) DNA. The aqueous phase of the droplet contained an ELP (Figure 10) and 0.1 pM F- Tar (-) DNA. Next, the goal was to image the progression of SA reactions immediately in droplet microenvironments, as it is an established method to study NPs and LLPS of IDPs12,36. Uniform, microfluidic-generated droplets were not made, as the reactions could have taken place during the time between droplet generation and microscopy since microfluidic droplet generation is much slower than generation of polydisperse droplets by manual shaking. The composition of the aqueous phase of each droplet contained the DNA strands and proteins is given in Table 4. Droplets are pipetted onto a glass slide mounted on the heating stage. Images were then acquired using brightfield and fluorescence acquisition modes at the desired temperatures, below (25°C) and above the LLPS transition temperature (45°C) at different times.

[0125] For SA reactions at 25°C, samples were prepared with F-Tar (-) and Q-Tar (+) with and without protein. As a negative control, samples were prepared substituting Q-Tar (+) with unlabeled Tar (+) to discard the possibility of a loss of fluorescence due to photobleaching. The fluorescence was tracked at 1,5,10, and 30 minutes. For SA reactions at 45°C, the fluorescence was tracked at 1 (25°C), 5 (45°C), 10 (45°C), 30 (45°C) and 40 (25°C) min. At 45°C, the ELPs undergo LLPS capturing NAs in their coacervate (Figure 11), and the spatial distribution of the fluorophores changes with respect to the images at 25°C. The initial fluorescence is dispersed all over the droplets, but above 45°C the fluorescence is concentrated into a protein coacervate smaller than the droplet. Hence the comparison becomes difficult, and it w as decided to add an image at 25°C after 40 minutes after resolubilizing the protein to compare to the initial image at 25°C.

[0126] Results and Discussion.

[0127] Thermodynamics of strand annealing of Tar(+) and Tar(-) oligoDNAs.

[0128] Hybridization of Tar(+) and Tar(-), two complementary' oligoDNA sequences is a good model reaction for the study of NA chaperones because they are each largely self- complementary and readily fold into unimolecular hairpin-like structures at room temperature. Modeling of these hairpin-like structures using NUPACK softw are (https: / / www.nupack.org / ) at 0. 1 mM Na+gives a free energy of the secondary' structures of -8 kcal / mol and -9 kcal / mol for Tar(+) and Tar(-), respectively. Self-folding of these complementary' oligomers results in a significant kinetic barrier for the bimolecular hybridization and formation of the thermodynamically favored duplex. Modeling using NUPACK software suggests that the change in Gibbs free energy of the secondary structure associated with the bimolecular hybridization of Tar(+) and Tar(-) at 25°C is significant (-60 kcal / mol). At 45°C, the free energy of unimolecular folding of Tar(+) and Tar(-) is substantially less (®-2 kcal / mol), but the thermodynamic driving force for bimolecular hybridization is still substantial (-50 kcal / mol), so the reaction is expected to proceed more readily because the steric constraints have been lessened. Proteins that function as NA chaperones are known to enhance the kinetics of this reaction, presumably by lessening unfavorable electrostatic and steric barriers to, and increasing local reactant concentrations for, bimolecular hybridization. Published studies have also proposed entropic mechanisms for the action of NA chaperones.

[0129] ELPs and their fusions: E3, E3.10, and E1-40.COR30 bind NAs with distinct affinities.

[0130] Three different NA-binding ELP-based proteins were engineered. Each appreciably bind NAs nonspecifically, but with distinct NA binding affinities and LLPS behaviors in water. The first, ELP E3, is a low complexity TDP with 8 lysines evenly interspersed between 80 ELP pentamer sequences that confer a total of 8 positive charges (Table 1) at neutral pH. Previous studies indicate that E3 does not bind appreciably with ssDNA in its soluble state38(i.e., T < Tt; presented again in Figure 6), but does bind and recruit ssDNA into its coacervate upon LLPS (T > Tt) via electrostatic interactions.13

[0131] Based on previous research on E3 and DNA binding, a new- protein was engineered with more robust NA binding behavior, denoted as E3.10, comprising E3 concatenated with two synergistic RNA binding domains from the FUS protein that work together to bind NAs promiscuously.32The two NA-binding domains of E3.10 fused at the C-terminus of E3 comprise a folded RNA recognition motif (RRM) and a disordered arginine glycine-rich (RGG) domain (see Table 2 for the sequence of E3. 10). As shown in the gel retardation assays presented in Figure 1A and IB, soluble E3. 10 (i.e., at T < Tt) can bind the DNA oligos used in this study. Below the Tt of E3.10, binding to 0. IpM Tar (+) is evident above 1 pM protein, and binding to 0. 1 pM dsDNA, denoted as Tar(+»-), is evident above 2.5 pM protein. According to structure prediction of the oligos by NUPACK (Figure 5),39individually, Tar (+) and Tar (-) self-hybridize to form hairpin structures at 25°C and 45°C. Thus the NA-binding that RRM and RGG confer to E3.10 is consistent with previous studies32,40,41reporting strong binding affinity of FUS RRM-RGG2 sequences to structured NA regions such as G-quadruplex, hairpins, stem-loops, or complex secondary structures.32Furthermore, RRM and RGG domains have been identified as contributors to RNA binding and chaperoning of NA annealing reactions.20

[0132] Table 2: Elastin-like polypeptide composition. This table describes the sequence composition for all the proteins used in this work.

[0133]

[0134] Table 2.1: Elastin-like polypeptide composition.

[0135] El -40 is a canonical ELP homopolymer (guest residue = V) with no inherent NA binding capacity. Simon et al. concatenated El-40 to an RNA-binding RGG domain to create an RNA binding fusion protein with well-defined LLPS behavior.12For this study, a NA- binding fusion was engineered and denoted as E1-40.COR30, comprising a 30 aaNA binding region derived from the HCV CP, fused at the C-terminus of El -40 to obtain a robust NA binding ELP (see Table 2 for sequence) with well-defined LLPS behavior. The HCV CP has proven to be an effective chaperone for Tar(+)*Tar(-) hybridization21-32As shown in Figure 1C and ID, soluble El-40. COR30 binds to the oligos used in this study; below its Tt, binding to 0. IpM Tar (+) is evident above 5 pM protein, and binding to 0.2 pM Tar(+)*Tar(-) (duplex) is evident above 10 pM protein. Apart from Tar ssDNA, HCV CP is known to bind highly structured RNAs.17,26,29

[0136] Soluble E3.10 and E1-40.COR30 chaperone double-strand annealing of Tar(-) and Tar(+).

[0137] The capacity of E3, E3.10, and El-40. COR30 to stimulate SA of two 56 nt complementary’ DNA oligos Tar(-) and Tar (+) (0.1 pM each) was investigated (Fig. 2A). First, the formation of 56 bp dsDNA was characterized in the presence of varying amounts of the model proteins by gel electrophoresis. In the absence of protein, no SA was observed after incubating the reaction at 25°C for over 10 min, while significant annealing was observed when samples were heated to 62°C for 10 min and then cooled to 4°C (Fig. 2B, Fig. 2C and 2D, lanes 3 and 4). The addition of E1-40.COR30 above 0.1 pM led to measurable duplex formation at 25°C, achieving near-complete annealing above 75pM (Fig. 2C, lane 12). The addition of E3.10, above O.luM, enhanced the annealing of Tar DNA, reaching its peak at lOpM (Fig. 2D, lane 13).

[0138] Annealing is generally favorable at high temperatures where NAs adopt a favorable conformation with more available nucleotides ready to be hybridized;42however, at 25°C, NAs are generally in a conformation that is not prone to duplex formation; hence you need another factor to overcome the kinetic barrier. According to the putative predicted structure of Tar (+) and Tar (-) (Fig. 2A, Fig. 5), at 25°C, the ssDNA is self-hybridized and completely folded, and has no available free nucleotides for annealing. While above 45°C, Tar DNA species maintain a complex folded structure but with more available free nucleotides for intermol ecul ar hybridization. Adding a protein chaperone at 25°C can bring the DNA into a conformation favorable for annealing. E3. 10 and El-40. COR30 proteins are positively charged, and that may lead to DNA-protein electrostatic interactions that may increase propensity’ for duplex formation. Previous studies have indicated that local charge screening may be the cause behind disordered NA chaperones. The polycationic IDPs may act as counterions that screen DNA negative charges, similar to the effect of salt on electrostatic interactions.33Moreover, other amino acids of the sequence may mediate a DNA conformational change or structure destabilization by unfolding some of the loops formed by Tar oligos.43,44This is consistent as amechanism of NA-binding of RRM-RGG domains or HCV CP is not exclusively electrostatic and can be involved in NA folding and unfolding events.17,22,45 A fluorescence quenching assay of SA was developed using a version of the Tar (-) oligo with a sequence modification in the first 5 nt (Table 3). This oligo, F-Tar(-), was labeled at the 3’ end with an ATTO488 fluorophore, and the Q-Tar (+) oligo was labeled at its 5’ end with a 5IABkFQ quencher (Figure 3A). These sequences are not entirely complementary as they were designed to have a free unhybridized toehold in each strand (see below). With this assay, the kinetics of the annealing reactions were quantified and tracked in real-time as the fluorescence from the F-Tar (-) strand is quenched upon the strand hybridization with the Q- Tar (+) strand. The concentrations of E3.10 and E1-40.COR30 that were selected for the fluorescence assay were the lowest protein concentrations with the highest annealing activity observed in the gel electrophoresis experiments (Figure 2). By selecting the lowest concentrations required for the maximum annealing activity, potential effects of molecular crowding were minimized. Soluble E3 does not bind appreciably to DNA. Thus, the assay was also performed with a very high E3 concentration (2 mM) to study the possible influence of molecular crow ding. Table 3: Description of DNA oligomers used in experiments. The sequences of the different oligo DNA species used in experiments are provided.

[0139] Consistent with the electrophoresis experiments, the fluorescence assays indicate that El-40. COR30 and E3.10 have chaperone activity in DNA strand annealing reactions at 25°C (Figure 3B). However, E3 has very little chaperone activity over 4 hours (Figure 3B), which is not surprising as E3 does not have measurable DNA binding activity' at this temperature (Figure 6). For El-40. COR30 and E3.10, the reaction is fast, reaching a steady level of fluorescence quenching within 30 min and reaching half of the final extent of reaction in under 5 min (Figure 3B). The fraction of DNA duplexed at room temperature after 4 hours is between 0.60 and 0.65 for E3. 10 and between 0.65 and 0.70 for E1-40.COR30 (Figure 3B). This seems slightly lower than what was observed by electrophoresis, but one has to consider that the sensitivity of detection of the experiments, quantification method, and the SA conditions are not the same in each experiment. While the gel electrophoresis assays used oligos that are complementary over all the 56 nts of each DNA strand, the oligos used in the fluorescence assays were designed to have a 5nt unhybridized toehold region for DNA strand displacement studies (see below). This difference in the sequence suggests that single-stranded F-Tar(-) is less folded at 25°C (Figure 5) with several unhybridized nts at both ends of the sequence. Q-Tar(+), however, has the same sequence as Tar (+) and should have a similar extent of folding in its single-stranded form. These factors may affect the dissimilarities between the extents of reactions observed in the electrophoresis and fluorescence readouts.

[0140] Fluorescence microscopy was used to examine the reactions depicted in Figure 3A and to ensure that complex coacervation did not occur within the aqueous solutions tested. The same reactants and chaperones were included into polydisperse aqueous microcompartments (droplets) in oil and observed their fluorescence over time under fluorescence microscopy. A negative control was introduced, substituting the quencher strand with an unlabeled Tar (+) strand to discard the possibility of photobleaching or fluorophore degradation as the reasons for fluorescence decays over time. Interestingly, for every' sample, significant loss in fluorescence was not observed in the negative controls (Figure 8, A-D), which indicates that all the decay in fluorescence is caused by quenching upon SA. Microscopy at 25°C shows no quenching in samples without protein (Figure 8. panels El-Hl) and minimal quenching of samples with E3 (Figure 8, panels E4-H4) over 30 min. El -40.COR30 exhibits SA chaperoning activity as the fluorescence signal becomes dimmer over time (Figure 8, E2-H2). The same is observed for E3. 10 (Figure 8, panels E3-H3), consistent with the findings above. No evidence of complex coacervation was observed at 25°C.

[0141] DNA SA is enhanced upon condensation into a protein-rich coacervates.

[0142] The LLPS transition temperature of the three model NA-binding ELPs in the presence and absence of DNA is presented in Fig. 4B & Fig. 9. Consistent with previous observations5 17addition of NAs lowers the LLPS transition temperature of NA-binding ELPs (Figure 4B and 9). Upon phase separation, NA-binding ELPs condense NAs into nucleoprotein coacerv ates that are thermodynamically stable and can be thermally reversible (Figure 10). There have been no previous studies of how two, individually folded, but complementary', DNA strands behave within such protein coacervates. It was assessed whether conformationally constrained DNA strands (i.e., F-Tar (-) and Q-Tar (+)) hybridize upon LLPS of the model ELPs by conducting the fluorescence-quenching assay at 45°C, above their Tts (Fig. 4A). It was observed that each of the proteins enhanced the formation of DNA duplexes. The raw fluorimetry data for each experiment are provided in Figure 7. The variances in the data for samples at 45°C are comparable to those at 25°C, suggesting that the plate reader measurements effectively captured fluorescence within the coacervates The reaction kinetics were significantly enhanced by protein LLPS at 45°C, with a 54-fold increase for E3. 10 and a 33-fold increase for E1-40.COR30 (Figure 4C), and a 3-fold increase for E3 (Figure 4C). all compared to the control reaction without protein (Figure 4C). These fold increases were calculated by comparing the half-times of the reactions, highlighting the substantial acceleration of kinetics in the presence of LLPS compared to the protein-free control.

[0143] 1.5mM of E3 and lOpM E3.10 were mixed as the protein chaperone (Figure 4C) to accelerate the reaction at similar rates as E3.10 only. As shown in Figures 9 and 10, the phase behavior of E3.10 is not the same as a canonical ELP. E3.10 phase separates and condensates DNA in small protein condensates that do not coalesce into a spherical liquid-like coacervate (Figure 10, panel B3). Moreover, after going back to room temperature, below the Tt of E3.10, solubilization of the coacervate remains incomplete, and DNA-protein complexes were observed (Figure 10, panel C3), which may be an indicator of non-reversible, aberrant phase separation as previously observed in FUS protein.52’53This can be due to unfolding of RRM domain due to temperature denaturation, which has been observ ed in irreversible self-assembly into irreversible amyloid fibers.48Adding E3 into the mixture results in the recovery of a spherical coacervate upon phase separation in which E3, E3-10, and DNA seems to be miscible (Figure 10. panel B4). However, upon going back to 25°C, the coacervate does not completely dissolve and evidence for the persistence of insoluble DNA complexes is observed (Figure 10, panel C4).

[0144] The SA reaction inside protein coacervates was observed as a function of time at 45°C using fluorescence microscopy. The same reactants used for the fluorimetry experiments were prepared in poly disperse droplets. A negative control was introduced by substituting the Q-Tar (+) strand with the unlabeled Tar (+) to confirm that the loss of fluorescence in the images was not due to degradation of the ATTO488, or to quenching by the coacervate. A fluorescence decay was not observed in the negative controls (Figure 11, panels A-E). Consistent with plate reader fluorimetry measurements, the sample without protein has a gradual fluorescence decay (Figure 1 1, panels Fl-Jl). For all protein-containing samples, fluorescence decay is observed rapidly after 5 minutes at 45°C (Figure 11, panels G2-G5). However, at 5 min, proteins have not formed fully coalesced condensates; hence at 10 mins (Figure 11, panels H2-H5), the fluorescence seems to be slightly higher. This is because, upon complete coalescence, all the DNA that emits the fluorescence signal is more concentrated in a smaller area, leading to a brighter zone in the droplet. Consequently, an image was added after protein resolubilization at 25°C (Figure 11, panels J2-J5) to compare with the initial state (Figure 11, panels F2-F5) and confirm that every sample is consistent with the fluorimetry data, and that the fluorescence signal decreases faster upon LLPS.

[0145] The microscopy images confirm that DNA is condensed within the protein coacervate immediately upon LLPS (Figures lOand 11) as previously observed,12’13and taken together with the fluorimetry data, it is concluded that upon LLPS, the SA reaction is faster in the coacerv ates above Tt than it is below Tt in the soluble state. Moreover, the reaction with phase separated ELPs is faster than the control without protein, indicating that reaction is not solely affected by temperature but also by the phase separation of the proteins. ELPs may play a role in the kinetics of such reactions by condensation of DNA strands into protein-rich coacervates and increasing the de facto concentration of the reactants. Charge screening by positively charged proteins may have a role by eliminating any electrostatic repulsion between DNA strands.33Moreover, upon LLPS the charge density may increase due to protein condensation, thus the charge screening effect may be higher than at 25°C. Considering that E3-DNA interactions are electrostatic, it is reasonable to think that the chaperoning of E3 is due to temperature, charge screening of repulsive DNA interactions33and DNA concentration in its coacervate. Nevertheless, other mechanisms may be involved for E3.10 and E1-40.COR30 since their reactions are faster than with E3 and their protein-NA interactions are not solely electrostatic. At 45°C ssDNA strand unfolds and becomes more thermodynamically favorable for a SA reaction.46 47However, it may be possible that upon LLPS at 45°C, not only is DNA is more prone to SA and more concentrated in the coacervate, but ELPs change their conformational structure which may result in different molecular interactions. This may fall into the idea of entropy transfer which suggests that the ordering of a chaperoning is complementary to the unfolding of a substrate.22

[0146] Conclusion.

[0147] This study demonstrates the use of engineered IDPs as potent NA chaperones for DNA annealing. The work uses ELPs as model IDPs with controllable phase behavior to explore new approaches to DNA chaperoning. This work provides evidence of the utility and versatility' of such proteins. Three different NA-binding ELPs with different structures, lengths, sequences, and mechanisms for DNA binding were used, and all of them demonstrated NA chaperone capability under different conditions. Inspired by previous studies and nature,12 13’17’25’32robust NA-binding ELPs were engineered that can interact wi th highly structured nucleic acid and can chaperone slow SA reactions below the Tt. ELP’s well-characterized LLPS behavior was also taken advantage of. The use of chaperoning SA reactions in less than 10 minutes upon recruiting the DNA species in phase-separated membrane-less condensates was demonstrated. The catalysis of such reactions in synthetic cells and nucleoprotein membrane-less organelles has not been previously explored. As the field of synthetic organelles grows in interest,49the discoveries presented herein are relevant due to the applications and impact on society’. DNA intermolecular hybridization reactions are at the heart of many molecular biology techniques. The catalysis of such reactions lead to the optimization of existing NA-based biotechnologies and will lead to the development of new biotechnologies.50

[0148] Bibliography

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[0153] 6. Dyson, H. J. Roles of intrinsic disorder in protein-nucleic acid interactions. Mol. BioSyst. 8, 97-104 (2012).

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[0158] 11. Meyer, D. E. & Chilkoti, A. Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat Biotechnol 17, 1112-1115 (1999).

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[0160] 13. Perez, T. D. et al. DNA Binding by an Intrinsically Disordered Elastin-like Polypeptide for Assembly of Phase Separated Nucleoprotein Coacervates. Ind. Eng. Chem. Res. 60, 17408- 17416 (2021).

[0161] 14. Felsenfeld, G. & Miles, H. T. The Physical and Chemical Properties of Nucleic Acids. Annu. Rev. Biochem. 36, 407-448 (1967).

[0162] 15. Zhang, W.-W. & Matlashewski, G. Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage mLeishmania. mSphere 4, e00408-I9 (2019). 16. Morey, M. et al. A glimpse into past, present, and future DNA sequencing. Molecular Genetics and Metabolism 110. 3-24 (2013).

[0163] 17. Cristofari, G. The hepatitis C virus Core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand RNA in vitro. Nucleic Acids Research 32, 2623- 2631 (2004).

[0164] 18. Sugiman-Marangos, S. N., Weiss, Y. M. & Junop, M. S. Mechanism for accurate, protein-assisted DNA annealing by Deinococcus radiodurans DdrB. Proc. Natl. Acad. Sci. U.S.A. 113, 4308-4313 (2016).

[0165] 19. Woodson, S. A., Panja, S. & Santiago-Frangos, A. Proteins That Chaperone RNA Regulation. Microbiol Spectr 6, 6.4.21 (2018).

[0166] 20. Meyer, A. et al. The RGG / RG motif of AUF1 isoform p45 is a key modulator of the protein’s RNA chaperone and RNA annealing activities. RNA Biology 16, 960-971 (2019).

[0167] 21. Mayer, O., Rajkowitsch, L., Lorenz, C., Konrat, R. & Schroeder, R. RNA chaperone activity and RNA-binding properties of the E. coli protein StpA. Nucleic Acids Research 35, 1257-1269 (2007).

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[0172] 26. Ivanyi-Nagy, R., Lavergne, J.-P., Gabus, C., Ficheux, D. & Darlix, J.-L. RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae. Nucleic Acids Research 36, 712-725 (2008).

[0173] 27. Vo, M.-N., Barany, G., Rouzina, I. & Musier-Forsyth, K. Mechanistic Studies of Mini- TAR RNA / DNA Annealing in the Absence and Presence of HIV-1 Nucleocapsid Protein. Journal of Molecular Biology’ 363, 244-261 (2006). 28. Wu, T., Heilman-Miller, S. L. & Levin, J. G. Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity’ of HIV-1 nucleocapsid protein. Nucleic Acids Research 35, 3974-3987 (2007).

[0174] 29. Ivanyi-Nagy, R. et al. Analysis of hepatitis C virus RNA dimerization and core-RNA interactions. Nucleic Acids Research 34, 2618-2633 (2006).

[0175] 30. Holmstrom, E. D., Liu, Z., Nettels, D., Best. R. B. & Schuler, B. Disordered RNA chaperones can enhance nucleic acid folding via local charge screening. Nat Commun 10, 2453 (2019).

[0176] 31. Sharma, K. kant et al. Kinetic analysis of the nucleic acid chaperone activity’ of the Hepatitis C virus core protein. Nucleic Acids Research 38, 3632-3642 (2010).

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[0178] 33. McDaniel, J. R., MacKay, J. A., Quiroz, F. G. & Chilkoti, A. Recursive Directional Ligation by Plasmid Reconstruction Allows Rapid and Seamless Cloning of Oligomeric Genes. Biomacromolecules 11, 944-952 (2010).

[0179] 34. MacEwan, S. R., Hassouneh, W. & Chilkoti, A. Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli. JoVE 51583 (2014) doi: 10.3791 / 51583.

[0180] 35. Fong. B. A., Wu, W.-Y. & Wood, D. W. Optimization of ELP-intein mediated protein purification by salt substitution. Protein Expression and Puri fication 66, 198-202 (2009).

[0181] 36. Simon, J. R., Carroll, N. J., Rubinstein, M., Chilkoti, A. & Lopez, G. P. Programming molecular self-assembly of intrinsically disordered proteins containing sequences of low complexity. Nature Chem 9, 509-515 (2017).

[0182] 37. Tsuchihashi, Z. & Brown, P. O. DNA strand exchange and selective DNA annealing promoted by the human immunodeficiency virus type 1 nucleocapsid protein. J Virol 68, 5863- 5870 (1994).

[0183] 38. Diez Perez, T. et al. Isolation of nucleic acids using liquid-liquid phase separation of pH-sensitive elastin-like polypeptides. Sci Rep 14, 10157 (2024).

[0184] 39. Zadeh, J. N. et al. NUPACK: Analysis and design of nucleic acid systems. J. Comput. Chem. 32, 170-173 (2011). 40. Loughlin, F. E. et al. The Solution Structure of FUS Bound to RNA Reveals a Bipartite Mode of RNA Recognition with Both Sequence and Shape Specificity. Molecular Cell 73, 490-504.e6 (2019).

[0185] 41. Hoell, J. I. et al. RNA targets of wild-type and mutant FET family proteins. Nat Struct Mol Biol 18, 1428-1431 (2011).

[0186] 42. Wong, K. L. & Liu, J. Factors and methods to modulate DNA hybridization kinetics. Biotechnol. J. 16, 2000338 (2021).

[0187] 43. Doetsch, M., Furtig, B., Gstrein, T., Stampfl, S. & Schroeder, R. The RNA annealing mechanism of the HIV-1 Tat peptide: conversion of the RNA into an annealing-competent conformation. Nucleic Acids Research 39, 4405-4418 (2011).

[0188] 44. Herschlag, D. RNA Chaperones and the RNA Folding Problem. Journal of Biological Chemistry 270, 20871-20874 (1995).

[0189] 45. Masuzawa, T. & Oyoshi, T. Roles of the RGG Domain and RNA Recognition Motif of Nucleolin in G-Quadruplex Stabilization. ACS Omega 5, 5202-5208 (2020).

[0190] 46. Taniue. K. & Akimitsu, N. Aberrant phase separation and cancer. The FEBS Journal 289, 17-39 (2022).

[0191] 47. Tmka, F. et al. Aberrant Phase Separation of FUS Leads to Lysosome Sequestering and Acidification. Front. Cell Dev. Biol. 9, 716919 (2021).

[0192] 48. Boczek, E. E. el al. HspB8 prevents aberrant phase transitions of FUS by chaperoning its folded RNA-binding domain. eLife 10. e69377 (2021).

[0193] 49. Staufer, O. et al. Building a community to engineer synthetic cells and organelles from the bottom-up. eLife 10, e73556 (2021).

[0194] 50. Mohammadniaei, M. et al. A non-enzymatic, isothermal strand displacement and amplification assay for rapid detection of SARS-CoV-2 RNA. Nat Commun 12, 5089 (2021).

[0195] Example II

[0196] Smart nucleic acid chaperones (SNACs) for toehold-mediated strand displacement (TMSD) reactions.

[0197] Toehold-mediated strand displacement (TMSD) is a significant enabling technology for many types of dynamic DNA nanoarchitectures. Here it is demonstrated that SNACs incorporating fusions of known NA chaperones and IDPs engineered to undergo triggered and reversible LLPS significantly accelerate TMSD reactions, both in a single-phase state (soluble) and in a concentrated SNAC-rich phase separated state. Table 5 provides the sequences of the ELPs and ELP fusion proteins investigated as potential SNACs for TMSD reactions. F-Tar (+) and Q-Tar (-) were designed to be complementary over 51 of their 56 nt sequence, leaving a 5nt toehold on each strand (Figure 12, Table 6). A 56nt oligo, denoted as 5nt-Tar (+) (Figure 12, Table 6), with 100% complementarity to F-Tar (-) was purchased to anneal to the fluorescent strand on the free 5nt toehold and displace the Q-Tar (+) quencher strand (Figure 12). Below the data from fluorescence spectroscopy and microscopy is presented to document the chaperone behavior of engineered proteins on TMSD reactions.

[0198]

[0199] Materials and Methods.

[0200] Assay of toehold mediated strand displacement.

[0201] 20 LIL solutions with equimolar DNA concentrations with and without protein were prepared as depicted in Table 7 by dilution into sodium phosphate (100 mM) buffer and loaded into a 384-well microtiter plate. Samples were first incubated for pre-annealing for 2 hr at 45°C. Evety 10 minutes, samples were excited at 488nm, and fluorescence intensity was measured at 520 nm at 45°C to check the annealing and subsequent quenching of the fluorophore was successful. 20pL solutions containing 0.1 pM dsDNA (F-Tar(-)»Q-Tar(+)) and 0.5 pM 5nt- Tar(+) with and without protein were prepared in triplicate, as noted in Table 7, and loaded into a 384-well microtiter plate. TMSD was characterized by a fluorescence recovery caused by displacement of the quencher-labeled strand Q-Tar(+). For 4 hours, every 2 minutes, samples were excited at 488nm, and fluorescence intensity was measured at 520 nm at 25°C and 45°C to study the reaction while the ELPs were in their soluble and phase separated states. Along with the TMSD reactions we measured with the same settings described above the fluorescence of dsDNA samples containing O. lpM F-Tar(-)«Q-Tar(+) as negative control, and samples containing 0.1 pM F-Tar(-)*5nt-Tar(+) as positive control, with and without protein. The raw fluorescence data is presented in Figure 14.

[0202] Table 7: Samples for SA and TMSD Reactions. Detailed sample composition for fluorimetry measurements and microscopy of SA and TMSD reactions for Figures 12 and

[0203] Fluorescence microscopy.

[0204] To image the process of the progression of the TMSD reactions in one and two-phase reaction systems as described above, an Olympus 1X83 fluorescence microscope (Olympus Life Science Technology Division, Center Valley, PA) equipped with a heating stage (TS4- MP / ER / PTU, Clifton, NJ) was used. For every sample, polydisperse microdroplets were prepared by mixing an aqueous phase and an oil phase in a microcentrifuge tube and brief manual shaking, our goal was to image the progression of TMSD reactions immediately in the droplet microenvironments, as it is an established method to study NPs and LLPS of IDPs.Rs Uniform, microfluidic-generated droplets were not made for this purpose as the reactions could have taken place during the time between droplet generation and microscopy since microfluidic droplet generation is much slower than polydisperse droplet generation by manual shaking. The aqueous phase of each droplet contained the DNA strands and proteins as presented in Table 7. Droplets are pipetted onto a glass slide mounted on the heating stage. Images were then acquired using brightfield and fluorescence acquisition modes at the desired temperatures, below (25°C) and above the LLPS transition temperature (45°C) at different times.

[0205] For TMSD reactions, Q-Tar (+) and F-Tar (-) were pre-annealed to quench the fluorescent signal of F-Tar (-) by heating at 45°C for 2hrs in the microplate reader measuring the fluorescence every ten minutes as done previously to control the annealing of the reaction. Afterward, solutions containing 0.1 pM F-Tar(-)»Q-Tar(+) and 0.5 pM 5nt-Tar(+) were prepared with and without protein. Also, a sample using added protein and 5nt-Xl was prepared as a noncomplementary negative control (Table 6). After a solution was made, poly disperse droplets were immediately made. The droplets were imaged at 1 (25°C), 5 (45°C), 10 (45°C), 30 (45°C), and 40 (25°C) minutes. A successful TMSD reaction was characterized by a recovery of fluorescence with respect to the initial state.

[0206] Results and Discussion.

[0207] An assay for toehold mediated strand displacement (TMSD) reaction and its catalysis by NA chaperones.

[0208] Two 56nt strands (F-Tar(-) and Q-Tar(+)) were designed that are have complementary sequences over 51nt and which also self-hybridize to form hairpin structures (Figure 13).1When the strands are annealed, they form a 51 bp dsDNA with two 5nt toeholds.2Adding a 56nt trigger with 100% complementarity' to either of the strands should displace one DNA strand to form a 56bp dsDNA. However, the displacement reaction is likely to be slow at ambient temperature because NUPACK predicts that the trigger strand also self-hybridizes (Figure 13, free energy of the secondary structure is -9 kcal / mol at 25°C and -2 kcal / mol at 45°C), indicating that the annealing from the toehold region would require significant unfolding at room temperature. The Gibbs free energy for this TMSD reaction, as estimated by NUPACK at 25°C is -4 kcal / mol; at 45°C it is similar, -3 kcal / mol. A fluorescence unquenching reaction was designed to study this TMSD process in the presence and absence of the model proteins (Figure 12). The assays consists of displacing the Q-Tar(+) strand from the formed dsDNA (F-Tar(-)*Q-Tar(+)) by the addition of 5nt-Tar(+) trigger. This TMSD reaction was observed by fluorescence spectrophotometry in a plate reader and by fluorescence microscopy of aqueous droplets containing the reactants and proteins.

[0209] ELPs chaperone TMSD reactions in solution and upon LLPS.

[0210] At 25°C, the trigger was added and samples without protein, with E1.40COR30, E3 or a mixture of E3+E3.10 showed no fluorescence recovery, indicating no TMSD activity (Fig. 14, columr " ’ i rast, samples with E3.10 exhibit a remarkable recovery in fluorescence, to over hah or me value measured for the unhybridized F-Tar(-) control (Figure 14, panel Cl). The IDP chaperones may change the configuration of DNA upon DNA binding, and these results can suggest that E3.10 has a more substantial effect on the conformation of DNA than E1.40COR30 as it has more versatility in its NA-binding domains. That is, E3.10 has RRM and RGG domains reported to disrupt dsDNA and RNA stem-loops.3,4TMSD reactions can be favored by unfolding the dsDNA structure and favoring the trigger invasion.5,6While E1.40COR30 does not catalyze TMSD reaction at 25°C, it catalyzes SA reactions by unfolding DNA hairpin structures. E3.10 may destabilize the dsDNA duplex and catalyze the invasion and annealing of the ttrigger 5nt-Tar(+) trigger simultaneously, while E1.40COR30 may not. Hence, E3.10 can chaperone this TMSD reaction at room temperature.

[0211] At 45°C, upon the addition of the trigger to samples without protein, no recovery in fluorescence was observed, indicating no significant displacement of the quencher strand by the trigger and additional annealing of the complementary strands even in the presence of the trigger (Figure 14, panel A2; Figure 15, panels F 1 -JI). This indicates that the reaction is either very slow or not possiblejust by this temperature stimulus. Destabilization of the folded trigger and / or the dsDNA may be necessary to favor the invasion of the trigger. The sample containing E3 exhibited a very minor increase in fluorescence upon the addition of the trigger, indicating some strand displacement (Figure Al 5, panel D2; Figure 15, panels F5-J5). TMSD is closely related to the process of SA. It can be catalyzed by disruption of dsDNA structures and annealing of the invader strand.7Considering that E3-DNA interactions are relatively weak and result in slow kinetics in SA reactions compared to the other model SNACs studied, the E3 coacervates may not contribute to the unfolding of dsDNA and that the small displacement observed may be due to the elevated temperature (45°C) and DNA concentration and / or screening of DNA repulsive charges in phase-separated condensates.8

[0212] Fluorescence recovery in samples containing E1.40.COR30 at 45°C occurred a few minutes after the trigger was added and the fluorescence reached a level commensurate with that of the unhybridized control (Figure 14, panel B2; Figure 15, panels F2-J2). Consequently, it may be that E1.40COR30 has the capacity to favor the invader upon LLPS by DNA concentration and possible conformational changes and entropic exchange to unfold ssDNA hairpin structures.9,10Note that SA happens with El 40COR30 at 25°C (see above), but TMSD does not. It may be that E1.40COR30 does not destabilize the dsDNA at 25°C, but it may be involved in such processes at higher temperatures upon protein / NA phase separation.

[0213] The fluorescence in E3. 10 samples increased soon after adding the trigger and reached levels commensurate with that of the unquenched control, indicating significant strand displacement (Figure 14, panel C2; Figure 15, panels F3-J3). In addition to previous results, this evidence adds to the idea that E3.10 has a strong influence on both ssDNA and dsDNA structures upon binding both in solution and in phase-separated states. As discussed above, the phase separation of E3.10 may form some fibers that are less accessible to the trigger even after manual resuspension of the sample. The last sample studied was the reaction in the presence of E3 and E3.10. As was observe, the mixture of E3 and E3. 10 do not show any fluorescence recovery. The addition of E3 may be interacting with E3.10 and interfering in some of the E3. 10-DNA interactions or that the presence of highly concentrated E3 increases the viscosity to the point of inhibiting the dynamics of the TMSD reaction.11

[0214] Bibliography

[0215] (1) Zadeh, J. N.; Steenberg, C. D.; Bois. J. S.; Wolfe, B. R.: Pierce. M. B.; Khan. A. R.; Dirks, R. M.; Pierce, N. A. NUPACK: Analysis and Design of Nucleic Acid Systems. J. Comput. Chem. 2011, 32 (1), 170-173.

[0216] (2) Zhang, D. Y .; Seelig, G. Dynamic DNA Nanotechnology Using Strand-Displacement Reactions. Nature Chem 2011, 3 (2), 103-113.

[0217] (3) Meyer, A.; Golbik. R. P.; Sanger, L.; Schmidt, T.: Behrens. S.-E.; Friedrich, S. The RGG / RG Motif of AUF1 Isoform P45 Is a Key Modulator of the Protein’s RNA Chaperone and RNA Annealing Activities. RNA Biology 2019, 16 (7), 960-971.

[0218] (4) Loughlin, F. E.; Lukavsky, P. J.; Kazeeva, T.; Reber, S.; Hock, E.-M.; Colombo, M.; Von Schroetter, C.; Pauli, P.; Clery, A.; Miihlemann, O.; Polymenidou. M.; Ruepp, M.-D.; Allain, F. H.-T. The Solution Structure of FUS Bound to RNA Reveals a Bipartite Mode of RNA Recognition with Both Sequence and Shape Specificity7. Molecular Cell 2019, 73 (3), 490-504.e6.

[0219] (5) Doetsch, M.; Schroeder, R.; Furtig, B. Transient RNA-Protein Interactions in RNA Folding: Transient RNA-Protein Interactions in RNA Folding. FEBS Journal 2011, 278 (10), 1634-1642. (6) Yong, X. E.; Palur, V. R.; Anand, G. S.; Wohland, T.; Sharma, K. K. Dengue Virus 2 Capsid Protein Chaperones the Strand Displacement of 5'-3' Cyclization Sequences. Nucleic Acids Research 2021, 49 (10), 5832-5844.

[0220] (7) Yong, X. E.; Palur, V. R.; Anand, G. S.; Wohland, T.; Sharma, K. K. Dengue Virus 2 Capsid Protein Chaperones the Strand Displacement of 5'-3' Cyclization Sequences. Nucleic Acids Research 2021, 49 (10), 5832-5844.

[0221] (8) Holmstrom, E. D.; Liu, Z.; Nettels, D.; Best, R. B.; Schuler, B. Disordered RNA Chaperones Can Enhance Nucleic Acid Folding via Local Charge Screening. Nat Commun 2019, 10 (1), 2453.

[0222] (9) Tompa. P.; Kovacs, D. Intrinsically Disordered Chaperones in Plants and AnimalsThis Paper Is One of a Selection of Papers Published in This Special Issue Entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and Has Undergone the Journal’s Usual Peer Review Process. Biochem. Cell Biol. 2010, 88 (2), 167-174.

[0223] (10) Cristofari, G. The Hepatitis C Virus Core Protein Is a Potent Nucleic Acid Chaperone That Directs Dimerization of the Viral (+) Strand RNA in Vitro. Nucleic Acids Research 2004, 32 (8), 2623-2631.

[0224] (11) Wong, K. L.; Liu, J. Factors and Methods to Modulate DNA Hybridization Kinetics. Biolechnol. J. 2021, 16 (11), 2000338.

[0225] Example III

[0226] Switchable SNACs: Removal of SNACs from Nano-architectures Assembled through Chaperoning.

[0227] In this example, it is demonstrated how SNACs in which binding to NA nanostructures can be togged on and off can enable the implementation of SNACs as nano-assemblers that can (i) be added to unhybridized (or partially hybridized) NA reactants to catalyze assembly to a thermodynamically favored hybridized architecture, and then (ii) be removed from solution to leave the assembled NA architecture primarily. This process requires switching of the SNAC from a state in which it binds NAs to one in which it does not bind NAs, for example by changing solvent conditions such as ionic strength or pH.1Because of the sensitive dependence of tendency for degree of NA hybridization to depend on ionic strength, focus here is on pH as an environmental trigger to switch SNAC binding propensity and thus allow SNAC removal from a solution after NA assembly has been achieved. This process takes advantage of the ability of SNACs to undergo temperature dependent liquid-liquid phase separation both in their chemical states in which they either bind, or do not bind, NAs. To do so, ELPs in which the guest residues comprise histidine was taken advantage of, which has a pKa at near neutral pH. Table 8 (below) contains the protein sequences studied, which include an uncharged ELP control (El -80), a SNAC demonstrated above to have chaperone properties in strand annealing and TMSD (E1.40COR30) and two histidine containing ELPs (H-20) and (H-24) with similar composition, but different length (molecular weight).

[0228] Table 8: Amino acid sequences of the elastin-like polypeptides and fusion proteins used in this study.

[0229] The histidine containing ELPs studied (H-20 and H-24) were initially synthesized by Chilkoti and coworkers, who showed that these ELPs exhibited both temperature and pH dependent LCST-type phase behavior.2These investigators did not examine the NA-binding nor chaperoning behavior of these proteins. In previous work, it was demonstrated that the binding between these ELPs to NAs (and subsequent release) is pH dependent, and can be exploited for the purpose of NA extraction from biological samples (e.g., nasopharyngeal samples for COVID tests).1In this work, it was demonstrate that such pH and temperature dependent ELPs can function as switchable SNACs, or nanoassemblers, which can be added to solutions of complementary NAs such that their assembly is catalyzed, and then can be rendered nonbinding to release the NA nanoarchitecture and be effectively removed from solution through LLPS.

[0230] Materials and Methods.

[0231] Buffers.

[0232] To reduce complications of the NA hybridization dependence on ionic strength buffers, two low ionic strength buffers (each with ionic strength -0.01M and each made using deionized water) were utilized. pH 6.5 buffer: 0.02 M KH2PO4, 0.01 M Na2HPO43pH 8.5 buffer: : 0.1 M Tns, 0.1 M HC1.3

[0233] Strand annealing assays via fluorescence quenching.

[0234] As described in Examples 1 and 2, Tar (-) was modified on the 3' end. by attaching a fluorophore 3ATTO488 at the 3’ end (IDT, Coralville, IA). The Tar (+) strand was labeled with a 5’ Iowa Black FQ quencher (5IABkFQ), and denoted as Q-Tar(+). 20 pL solutions with equimolar DNA concentrations with and without protein were prepared in triplicate, as noted and loaded into a 384- well microtiter plate. Annealing was characterized by a fluorescence decay caused by quenching of ATTO488 by the proximity of 5IABkFQ. For 4 hours, every 2 minutes, samples were excited at 488nm, and fluorescence intensity was measured at 520 nm at 25°C.

[0235] Strand annealing assays via agarose gel electrophoresis.

[0236] Tar(+) and Tar(-) were diluted from IpM stock solution (IxTris / EDTA (TE), pH 7.5. 20pL annealing solutions containing equimolar 0.1 pM concentrations of DNA were prepared with and without protein, at two different pHs, as tabulated in Table 2 (below). All reactions, except 3 and 10, were incubated at room temperature for 10 min. Reactions 3 and 10 w ere incubated at 62°C for 10 min. Reactions were stopped by adding 10 pL of a solution containing 20% glycerol, 20 mM EDTA pH 8.0, 0.2% SDS, 0.25 % bromophenol blue. This solution denatures the protein and releases it from the DNA oligos. The extent of reactions w as resolved in 2.5% agarose gels in IX TBE at 4°C for 90 min at 65V. The gel is post-stained with SyBr Gold and imaged in a transilluminator.

[0237] Table 9: Compositions of reactants, H-24 SNACs and pH used for gel electrophoresis assays.

[0238] Chaperoning of NA annealing, subsequent release, and removal of switchable

[0239] SNAC.

[0240] Fig. 16 provides an overview of the experimental workflow for demonstration that H- 24, a switchable SNAC, can be released from a NA nano-assembly after it chaperones its formation.

[0241] Reaction mixtures (70 pL) containing equimolar concentrations (0.1 pM) of complementary oligo DNAs and 0.5 mM H24 protein were prepared in triplicate, as specified in Table 10 (below). To monitor DNA-protein annealing, 20 pL of each mixture was loaded into individual wells of a 384-well microtiter plate. Fluorescence quenching was measured as ATTO488 came into close proximity of the 5IABkFQ quencher upon intermolecular strand hybridization. Samples were excited at 488 nm and fluorescence intensity was recorded at 520 nm ever}72 minutes for 30 minutes at 25°C. Fluorescence decay was indicative of the annealing process between DNA as it is chaperoned by the H24 SNAC.

[0242] Table 10

[0243] The goal in preparing these samples was to track DNA fluorescence during the process of protein and DNA separation. To accurately assess DNA localization in the supernatant and coacervate phases following LLPS, control samples were prepared using both single-stranded (ssDNA) and double-stranded DNA (dsDNA). These controls allow monitoring of the behaviour of DNA under fluorescent measurements.

[0244] Results and Discussion.

[0245] Tar(+) and Tar(-) are stable hairpins in low ionic strength buffers.

[0246] Fig. 17 presents the extent of reaction (as defined in Example 1) for the room temperature hybridization of Tar(+) and Tar(-) (0.1 pM each) in the two low ionic strength buffers prepared (pH 6.5 and 8.5). At each pH, the extent of duplex formation remains low over the course of 4 hrs, indicating a significant activation energy barrier for the formation of the thermodynamically favored bimolecular duplex for these constrained, folded (i.e., selfhybridized) oligomers. Modeling of the hybridization reaction using the lowest salt concentration that NUPACK software allows (0.05 M) suggests that the Gibbs free energy of this reaction is significant (-59 kcal / mol, see Fig. 17).

[0247] As controls for the examination of pH-switchable SNACs, the effect of ELPs that should not interact chemically with DNA (El-80)4and a SNAC examined in Examples 1 and 2 (E140COR30) were examined, according to calculations conducted on SnapGene® softw are (from Dotmatics; available at snapgene.com). El-80 has only a slight charge at each pH examined (pH 6.5: +0.9; pH 8.5: +0.1; pl = 8.8), while E1.40COR30 is positively charged at each pH examined. (pH 6.5: +7.9; pH 8.5: +7.1; pl = 11.9). Fig. 18 summarizes the results of fluorescence quenching assays for bimolecular strand hybridization of Tar(+) and Tar(-) at room temperature in the presence of El-80 and E1.40COR30. In each case, increased levels of strand annealing were observed upon the addition of protein.

[0248] Addition of El-80 to the NA reaction mixture resulted in only modest increases in the hybridization reaction rate, reaching a maximum extent of reaction of approximately 0.6 after 4 hrs at pH 8.5 the highest El-80 concentration investigated (0.5 mM). No consistent effect of pH over the El -80 concentrations studied was observed. The rate of reaction (change in extent of reaction over time) did not correlate monotonically with El-80 concentration, with the slowest rate being observed for all pHs at 0.05 mM El-80. It may be that there are competing effects on the rate of reaction responsible for the trends observed. For example, it is known that molecular crowding effects upon the addition of soluble polymers that do not interact with NAs can enhance their hybridization efficiency through purely entropic effects.5While El-80 is only slightly charged at the pHs of interest, it does have a slight positive charge as noted above. Thus, addition of El -80 may also perturb the ionic balance of the solutions to have a secondary effect on hybridization rate beyond molecular crowding.

[0249] Addition of E1.40COR30 to the NA reaction mixture resulted in significant increases in the hybridization reaction rate, which reached a maximum extent of reaction of over 0.9 after 4 hrs at the highest E1.40COR30 concentration (0.5 mM) for all pH levels. This is consistent with the SNAC properties of E1.40COR30 observed in Examples 1 and 2 above and is likely due to the significant positive charge on this SNAC. As with El -80, the lowest rates of reaction were seen at the intermediate E1.40COR30 concentration investigated (0.05 mM). This again suggests competing processes at play upon increase of the concentration of this protein in the reaction mixture. As with El-80, no consistent effect of pH over the E1.40COR30 concentrations studied was observed. In the case of El .40COR30, another factor that may come into play is the stoichiometries of the binding between protein and the NAs and their complexes. pH-responsive histidine-containing ELPs are SNACs for DNA strand annealing below their pl.

[0250] The histidine-containing ELPs studied here have a high positive charge at pH 6.5 (H- 24: +27.3 (pl = 8.33), H-20: +22.7 (pl = 8.30), as estimated by SnapGene) and are almost neutral at pH 8.5 (H-24: -0.3. H-20: -0.4). It was thus hypothesized that they would be effective as SNACs at pH 6.5, but not at pH 8.5. Fig. 19 shows the results of fluorescence quenching assays for bimolecular strand hybridization of Tar(+) and Tar(-) at room temperature in the presence of increasing concentrations of H-24 at both pH 6.5 and 8.5. In each case, substantial differences in strand annealing kinetics and extent of reaction were observed at pH 6.5 and pH 8.5.

[0251] Addition of the highly charged H-24 at pH 6.5 resulted in dramatic increases in the hybridization reaction rate, to the point where the transient from low to high extent of reaction could not be captured within the time resolution of our fluorescence quenching assay because of the time lag between mixing of the H-24 and the oligo NA reagents and the commencement of fluorescent measurements in the plate reader (estimated to be a minimum of ~I min). The highest extent of reaction («1.0) was measured at 50 pM H-24, the highest concentration investigated; addition of 5 pM H-24 resulted in an extent of reaction of ®0.9, while addition of 500 nM H-24 resulted in a maximum extent of reaction of «0.8.

[0252] At pH 8.5, addition of H-24 resulted in some degree of annealing at all concentrations, but at a slow reaction rate compared to the annealing observed at pH 6.5. It is notable that at low reaction times, very dramatic differences in extent of reaction can be obtained in the presence and absence of the H-24 SNAC by varying the pH (e.g. ~I.O vs. ~0 at pH 6.5 vs pH 8.5). This indicates that significant optimization of reaction conditions can be employed to achieve a hybridization reaction of interest by toggling SNAC activity on and off by pH modulation.

[0253] Figure 20A provides the results of fluorescence quenching assays for bimolecular strand hybridization of Tar(+) and Tar(-) at room temperature in the presence of 500 nM H-20 at both pH 6.5 and 8.5. This histidine containing ELP also functions as an effective NA chaperone at pH 6.5 with the transient response for the extent of reaction again too fast to be resolved by our assay method. At pH 6.5, a steady state extent of hybridization of « 0.7 is maintained over the course of the 4 hr experiment. At pH 8.5, the hybridization remains very slow.

[0254] Fig. 20B shows the predicted molecular charge on the histidine-ELPs studied as a function of pH. The results obtained with the fluorescence quenching assay of intermolecular hybridization are correlated with the predicted molecular charges. At pH 8.5, both H-24 and H-20 have very low molecular charge, -0.4 and -0.3, respectively. At pH 6.5, both are predicted to have significant positive charge with the ratio of molecular charge (H-20 / H24: +22.7 / +27.3 = 0.83) roughly equivalent to ratio of the maximum extent of reactions observed in our comparable bimolecular hybridization assays (0.7 / 0.8 = 0.88). These results give significant insight on how to design effective SNACs at the molecular level.

[0255] In total, the results from the fluorescence quenching assays show that H-24 and H-20 are very efficient SNACs at pH 6.5. At pH 6.5, H-24 at 50 pM chaperones complete hybridization of 0.1 pM Tar(+) with 0.1 pM Tar(-) within the time resolution of our assay (~1 min). Over the same reaction time scales at pH 8.5, H-24 and H-20 do not promote significant levels of strand hybridization, presumably because they does not interact significantly with the oligoNAs.1These results are corroborated by the results of gel electrophoresis assays presented below.

[0256] Figure 21 shows the results of intermolecular hybridization reactions of Tar(+) and Tar (-) after 10 min reaction times as characterized by gel electrophoresis. Hybridized (annealed) duplexes show a characteristic slower mobility than unhybridized oligo NAs. Addition of as little as 10 pM H-24 at pH 6.5 to the NA reaction mixture results in significant strand annealing (i.e., formation of the hybridized duplex); increasing the concentration of H-24 to levels above 100 pM at pH 6.5 seems to result in a decrease in the degree of annealing (i.e. extent of reaction). At pH 8.5, no effect of addition of H-24 is apparent. These results are qualitatively consistent with those obtained from our fluorescence quenching assays.

[0257] These findings suggest that one can add these histidine containing ELPs to NA reaction mixtures at pH 6.5, to allow them to rapidly chaperone NA assembly, and then to switch the pH to a level in which they dissociate from the NAs, and can further be largely removed from the reaction solution through triggered phase separation. This process is demonstrated below. pH-dependent recruitment of DNA into H24 coacervates.

[0258] Triplicates of the mixtures (100 pL) containing 0.5 mM H-24 and either 0. 1 pM ssDNA (fluorescently labelled Tar(-) denoted F-Tar(-)) or 0.1 pM dsDNA (duplexed F-Tar) / / Tar (+)) were prepared at two different pH levels. The aim w as to examine whether H-24 can recruit ssDNA and dsDNA upon LLPS. At pH 6.5, H-24 is positively charged and readily binds DNA, while at pH 8.5, it becomes slightly negatively charged and does not bind ssDNA or ssRNA.1’6,7As needed to monitor pH-dependent DNA-protein binding, 20 pL of each mixture was loaded into individual wells of a 384-well microtiter plate. LLPS was then induced in all samples by heating to 65°C for 10 min, followed by 1 min of hot centrifugation. The supernatant (proteinpoor phase) was carefully decanted, and the coacervate (protein-rich phase) was resuspended using the same buffer. Both phases were subsequently measured for fluorescence intensity and compared to the original buffer to assess the presence of DNA in each phase.

[0259] As shown in Fig. 22, at pH 6.5. both ssDNA and dsDNA were predominantly localized within the H-24 coacervate after LLPS, confirming that the system is in the DNA-binding regime. This is to be expected as H-24 has a high positive charge at this pH, enabling significant electrostatic interactions with the negatively charged DNA. In contrast, at pH 8.5, ssDNA and dsDNA were found in the supernatant, indicating that DNA and protein separated following LLPS. This confirms that at higher pH, when H-24 is slightly negatively charged, it does not bind ssDNA nor dsDNA appreciably, preventing DNA from associating with the coacervate.

[0260] These results demonstrate that H-24 can effectively sequester ss or ds DNA upon LLPS at pH 6.5, and suggests, conversely, that DNA can be released DNA from a coacervate formed at 6.5 when it is resuspended at higher pH. This hypothesis is further probed in the experiments detailed below, where the goal is to separate DNA assembly reaction products from a proteinbased NA chaperone following its successful facilitation of DNA annealing reactions.

[0261] Histidine-containing ELPs are pH-switchable SNACs that can be effectively removed by LLPS from NA nanoarchitectures after their assembly.

[0262] To demonstrate that utility of triggered LLPS in removing pH switchable SNACs from reaction mixtures, the protocol summarized in Fig. 1 , and its associated text, was followed. The Tar(+), Tar(-) annealing reaction w as repeated in the presence of 0.5 mM H-24. This higher concentration of H-24 was used to facilitate formation of a good amount of protein-rich phase upon phase separation so that the protein-poor phase could be easily decanted. To enable tracking of the segregation and release of NAs from the coacervated phases, control experiments w ere performed with just the fluorescently labeled oligoNA (F-Tar(-)) and with mixtures of the F-Tar(-) and Tar(+) not labeled with the quencher. If only reactions in which oligo DNAs were labelled with a fluorophore and quencher were used, it would have been difficult to definitively determine DNA localization because the lack of fluorescence could be attributed to either quenching of the fluorophore upon annealing or the absence of DNA. Since these two phenomena are indistinguishable in this context, parallel controls were prepared lacking the quencher to provide fluorescent measurements without significant quenching. The experimental conditions for the three reactions are given in Table 3.

[0263] Fluorescence measurements of the extent of annealing reaction between fluorophore- and quencher-labelled oligo DNAs were consistent with the results of Figs. 19 and 21, indicating that H-24 effectively chaperones strand annealing at 25°C and pH 6.5 (extent of reaction after 30 min «0.9). No extent of reaction was obtained in the fluorescence assay for the control reactions in Table 3 because of the absence of the quencher-labelled strand.

[0264] 20 pL was recovered from the microtiter plate used to measure the extent of strand annealing reaction and added it back to the original reactions of 70 pL. To shift the pH, 1.2 pL of NaOH was added to each 70 pL tube of the DNA-protein mixtures, and the samples were thoroughly mixed. LLPS was then induced by heating the samples to 60°C for 10 minutes, followed by a 1 -minute hot spin centrifugation. After phase separation, the supernatant was decanted, and the remaining coacervate phase was resuspended to the original volume with a pH 6.5 buffer. Both phases were subsequently measured for fluorescence intensity and compared to the original buffer to assess the presence of DNA in each phase. From each phase (supernatant and coacervate). 20pL was transferred to a 384-well microtiter plate for fluorescence measurement. Fluorescence intensity' was measured once at 25°C, with excitation at 488 nm and emission recorded at 520 nm.

[0265] The fluorescence of the protein-rich and protein-poor phases of reactions 1 and 3 were measured and compared to the initial fluorescence right after the samples were prepared. The majority of the DNA localized in the protein-poor phase following LLPS at pH 8.5 (FIG. 23). Reaction 2 did not provide a reliable indicator for this measurement, as the absence of fluorescence could result either from the annealing of the fluorophore and quencher or from the actual absence of DNA. Therefore, it was not included in measurements for reaction 2, and thus, conducted control reactions 1 and 3 without the quencher.

[0266] To assess the localization of DNA and protein in each phase, 20 pL of the remaining samples from the supernatant and coacervate phases were used for both DNA and protein gel electrophoresis. For DNA analysis, reactions were stopped by adding 10 pL of a solution containing 20% glycerol, 20 mM EDTA (pH 8.0), 0.2% SDS, and 0.25% bromophenol blue to denature the proteins and release the oligoDNA. DNA samples were resolved using 2.5% agarose gels in IX TBE buffer at 4°C for 60 minutes at 75V. Gels were post-stained with SyBr Gold and visualized using a transilluminator (See Fig. 24A). For protein localization, 20 pL of the reaction mixtures were loaded into standard SDS-PAGE gels for electrophoretic separation (Fig. 24B). SDS-PAGE conditions and settings were applied according to standard protocols.8

[0267] The DNA gel analysis revealed that DNA was primarily localized in the protein-poor phase at pH 8.5, indicating that DNA is not recruited into the protein-rich coacervate after LLPS at this pH (Fig. 24A). This confirms that at higher pH, DNA and protein can be effectively partitioned into distinct phases.

[0268] H24 exhibits lower critical solution temperature (LCST) behaviour, becoming turbid when heated above its transition temperature. A simple turbidity test showed that the proteinpoor phase remains clear upon heating, indicating the absence (or minimal presence) of protein. In contrast, the protein-rich phase becomes turbid, consistent with the expected LCST behaviour (data not shown). This visual assessment was further confirmed by protein gel analysis (Fig. 24B), where the protein-rich phases exhibited strong protein bands compared to the H24 control, while the protein-poor phases did not produce a noticeable band.

[0269] These results, along with fluorimetry measurements, demonstrate that SNACs and the DNA reactants and products they act on can be successfully separated through a pH shift and subsequent LLPS. Specifically, H-24 can act as an effective chaperone for DNA annealing reactions in its charged state, and then the chaperone can easily be removed from the reaction mixture with a simple pH shift and LLPS.

[0270] Conclusions

[0271] Switchable SNACs are catalysts that can facilitate and enable kinetically trapped NA hybridization reactions, including strand annealing, toehold mediated strand displacement, and other useful supramolecular assembly processes in NA nanotechnology'. Upon completion of the desired reactions, the switchable SNACs can be transformed into a state that does not interact with the reaction products and can be effectively separated from the reaction products by triggered phase separation. The reaction components and conditions presented here represent a first demonstration of switchable SNACs, many other extensions can be envisioned including employment of different NA reagents, employment of other SNACs that are switchable between NA chaperoning and nonbinding states, employment of more efficient means of LLPS and phase and product recovery' besides decanting of supernatant (e.g., filtration, electrophoresis).

[0272] Bibliography 1. Diez Perez, T. et al. Isolation of nucleic acids using liquid-liquid phase separation of pH-sensitive elastin-like polypeptides. Sci Rep 14, 10157 (2024).

[0273] 2. MacKay, J. A., Callahan, D. J., FitzGerald, K. N. & Chilkoti, A. Quantitative Model of the Phase Behavior of Recombinant pH-Responsive Elastin-Like Polypeptides.

[0274] Biomacromolecules 11, 2873-2879 (2010).

[0275] 3. Perrin, D. Buffers of Low Ionic Strength for Spectrophotometric pK Determinations. Aust. J. Chem. 16, 572 (1963).

[0276] 4. Simon, J. R., Eghtesadi, S. A., Dzuricky, M., You, L. & Chilkoti, A. Engineered Ribonucleoprotein Granules Inhibit Translation in Protocells. Molecular Cell 75, 66-75. e5 (2019).

[0277] 5. Hong, F.. Schreck, J. S. & Sulc, P. Understanding DNA interactions in crowded environments with a coarse-grained model. Nucleic Acids Research 48, 10726-10738 (2020).

[0278] 6. Ballin, J. D. et al. Contributions of the Histidine Side Chain and the N-Terminal a- Amino Group to the Binding Thermodynamics of Oligopeptides to Nucleic Acids as a Function of pH. Biochemistry 49, 2018-2030 (2010).

[0279] 7. Eliseo, T. et al. Indirect DNA Readout on the Protein Side: Coupling between Histidine Protonation, Global Structural Cooperativity, Dynamics, and DNA Binding of the Human Papillomavirus Type 16 E2C Domain. Journal of Molecular Biology 388, 327-344 (2009).

[0280] 8. Gallagher, S. R. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE). CP Essential Lab Tech 6, (2012).

[0281] The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the aspects of the present invention. Thus, it should be understood that although the present invention has been specifically disclosed by specific aspects and optional features, modification and variation of the concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of aspects of the present invention. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that the definition of a term incorporated by reference conflicts \\ i th a term defined herein, this specification shall control.

Claims

WHAT IS CLAIMED IS:

1. A method to increase nucleic acid hybridization comprising: a) contacting complementary nucleic acid strands with at least one smart nucleic acid chaperone (SNAC), wherein the SNAC comprises a nucleic acid chaperone polymer that undergoes liquid-liquid phase separation (LLPS); b) allowing the at least one SNAC to form a coacervate, encapsulate nucleic acids and chaperone hybridization of the complementary strands, wherein hybridization kinetics are increased as compared to hybridization kinetics without the presence of the at least one SNAC.

2. The method of claim 1, wherein the polymer comprises a synthetic polymer, elastinlike polypeptide (ELP), elastin, resilin, RGG, and HCV core protein (HCV CP), or a combination thereof.

3. The method of claim 2, wherein the ELP comprises repetitive sequences of V-P-G-X- G (SEQ ID NO: 1), where X is any amino acid except proline.

4. The method of claim 2, wherein the ELP comprises SEQ ID NO: 11 (E3), SEQ ID NO: 16; SEQ ID NO: 30 (H24), a combination thereof or 95% identity thereto.

5. The method of claim 1, wherein the SNAC comprises a nucleic acid binding domain, a viral capsid protein or combination thereof.

6. The method of claim 5, wherein the nucleic acid binding domain comprises an RNA recognition motif (RRM), an arginine-glycine (RGG) domain, a viral capsid protein comprising a hepatitis C virus core protein (HCV CP) or combination thereof.

7. The composition of claim 1, wherein the complementary nucleic acid strands comprise self-complementary regions that form hairpin structures.

8. The composition of claim 1, wherein the complementary nucleic acid strands comprise toehold regions for strand displacement reactions.

9. The method of claim 1, wherein b) further comprises inducing liquid-liquid phase separation (LLPS) of the SNAC to form polymer-rich coacervates that concentrate the nucleic acid strands, wherein the polymer-rich coacervates form nucleoprotein-like condensates that speed up hybridization kinetics.

10. The method of claim 9, wherein the polymer-rich coacervates enhance the rate of hybridization up to about 3- to about 60-fold compared to hybridization without coacervates.

11. A method to increase hybridization of nucleic acids comprising: a) contacting complementary nucleic acid strands with at least one pH-switchable smart nucleic acid chaperone (SNAC) polymer at a first pH, wherein the SNAC comprises a nucleic acid chaperone polymer that undergoes liquid-liquid phase separation (LLPS); b) allowing the at least one SNAC to form a coacervate, encapsulate nucleic acids and chaperone hybridization of the complementary nucleic acid strands of a); and c) after b) adjusting to a second pH to release the at least one SNAC from the hybridized nucleic acids. wherein hybridization kinetics are increased as compared to hybridization kinetics without the presence of the at least one SNAC.

12. The method of claim 11, wherein the at least one SNAC comprises SEQ ID NO: 29, SEQ ID NO: 30 or 95% identity thereto.

13. The method of claim 11, wherein the first pH is below the isoelectric point and the second pH is above the isoelectric point of the SNAC.

14. The method of claim 11, wherein the first pH is about 6 to 8 and the second pH is about 8.5 to 9.0.

15. The method of claim 11, wherein in b) SNAC coacer ates are formed in which the SNAC chaperones hybridization of the complementary nucleic acid strands within the coacervates, wherein the protein / nucleic acid-containing coacervates form nucleoprotein condensates that speed up hybridization kinetics.

16. The method of claim 11, wherein in c) the assembled components from the coacervates of b) are released and the chaperoning activity is disrupted, and the chaperone and nucleic acid product are separated.

17. The method of claim 11, wherein the complementary nucleic acid strands comprise self-complementary regions that form hairpin structures.

18. The method of claim 11, wherein the complementary nucleic acid strands comprise toehold regions for strand displacement reactions.

19. The method of claim 15, wherein the coacervates enhance the rate of hybridization up to about 3- to about 60-fold compared to hybridization without coacervates.

20. A method to increase hybridization of nucleic acids comprising: a) contacting complementary nucleic acid strands with at least one smart nucleic acid chaperone (SNAC) polymer at a first temperature, wherein the SNAC comprises a nucleic acid chaperone polymer that undergoes liquid-liquid phase separation (LLPS); b) allowing the SNAC to form a coacervate, encapsulate nucleic acids and chaperone hybridization of the complementary nucleic acid strands; and c) adjusting to a second temperature to dissolve the coacervate and release the SNAC from the hybridized nucleic acids, wherein hybridization kinetics are increased as compared to hybridization kinetics without the presence of the at least one SNAC.

21. The method of claim 20, wherein the first temperature is above the SNAC transition temperature, and the second temperature is below the SNAC transition temperature.

22. The method of claim 20, wherein in b) polymer / nucleic acid-containing coacervates are formed in which the SNAC chaperones hybridization of the complementary nucleic acid strands within the coacervates, wherein the polymer / nucleic acid-containing coacervates form nucleoprotein-like condensates that speed up hybridization kinetics.

23. The method of claim 22, wherein liquid-liquid phase separation (LLPS) is induced at the first temperature to form the coacervates and enhance hybridization.

24. The method of claim 22, wherein in c) the assembled components from the coacervates of are released and the chaperoning activity is disrupted, and the chaperone and nucleic acid product are separated.

25. The method of claim 24, wherein the temperature is lowered to dissolve the coacervates to release the hybridized products.

26. The method of claim 20, wherein the polymer comprises a synthetic polymer, elastinlike polypeptide (ELP), elastin, resilin, RRM-RGG, and HCV core protein (HCV CP), or a combination thereof.

27. The method of claim 26. wherein the ELP comprises SEQ ID NO: 11 (E3). SEQ ID NO: 16, SEQ ID NO: 30 (H24), a combination thereof or 95% identity thereto.

28. The composition of claim 20, wherein the complementary nucleic acid strands comprise self-complementary regions that form hairpin structures.

29. The composition of claim 20, wherein the complementary nucleic acid strands comprise toehold regions for strand displacement reactions.

30. The method of claim 22, wherein the polymer-rich coacervates enhance the rate of hybridization up to about 3- to about 60-fold compared to hybridization without coacervates.

31. The method of claim 11, wherein the polymer comprises a synthetic polymer, elastinlike polypeptide (ELP), elastin, resilin, RGG, and HCV core protein (HCV CP), or a combination thereof.

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